Antimicrobial agents and their use in therapy

ABSTRACT

The present invention provides an agent, or a composition containing an agent, for use in treating or preventing a bacterial infection in a subject, wherein said agent comprises:
         (i) an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound,
 
wherein the PCNA interacting motif is X 1 X 2 X 3 X 4 X 5  (SEQ ID NO: 1) and
 
wherein:
   X 1  is a basic amino acid;   X 2  is an aromatic amino acid;   X 3  is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);   X 4  is any amino acid other than Proline (P), an acidic amino acid or an aromatic amino acid; and   X 5  is a basic amino acid or Proline (P); or   (ii) a nucleic acid molecule comprising a sequence encoding the oligopeptidic compound of (i).       

     In certain aspects the agent and compositions of the invention may be used as single agents. In other aspects of the invention agents and compositions of the invention may be used in conjunction with one or more additional active agents, such as antibiotics.

The present invention relates to novel agents, particularly peptides ormimetics thereof and their encoding nucleic acids, pharmaceuticalcompositions comprising at least one of said agents, and their use asanti-microbials, e.g. in the treatment or prevention of microbialinfections, particularly bacterial infections. The agents may be usefulalone or in combination with other compounds, such as cytotoxic and/orcytostatic compounds, particularly intracellularly-acting cytotoxicand/or cytostatic compounds, including e.g. anti-microbials such asantibiotics, anti-fungals etc. Also provided are therapeutic methodswhich comprise the use of said agents and compositions for theaforementioned uses. The agents may also be used in the manufacture orpreparation of medicaments for the aforementioned therapies.Furthermore, the agents may be used in in vitro methods, e.g. in cellculture methods (to prevent or inhibit microbial growth or to prevent orreduce unwanted microbial colonisation or contamination in a non-medical(e.g. in vitro or ex vivo) setting, for example for sterilisation orantiseptic purposes) and in the production of products coated with theagent (e.g. medical devices, implants etc). Thus, products coated withthe agent are also provided.

APIM peptides are a group of peptides that interact with PCNA(proliferating cell nuclear antigen) via a novel PCNA interacting motif(Gilljam et al., 2009. Identification of a novel, widespread, andfunctionally important PCNA-binding motif, J. Cell Biol. 186(5), pp.645-654). The motif has been termed APIM (AlkB homologue 2 (hABH2)PCNA-interacting motif) since it was first identified as mediating theinteraction between hABH2 and PCNA, but as will be clear from thedisclosure below, APIM motifs have now been identified in a wide rangeof proteins. The, PCNA binding motif found in APIM peptides typically isdefined using the consensus sequence,[R/K]-[F/W/Y]-[L/I/V/A]-[L/I/V/A]-[K/R] (SEQ ID NO: 19).

PCNA is a member of the sliding clamp family of proteins, which is knownto be involved in both DNA replication and DNA repair. The main functionof PCNA is to provide replicative polymerases with the high processivityneeded for duplication of the genome. In live S-phase cells, PCNA taggedwith green fluorescent protein (GFP) forms distinct foci representingsites of replication. It can therefore be used as an S-phase marker.

Numerous proteins involved in cellular processes such as DNA repair,chromatin assembly, epigenetic and chromatin remodelling,sister-chromatid cohesion, cell cycle control and survival are localisedin so-called replication factories which contain more than a dozenreplication forks. Many of these proteins interact with PCNA and havebeen shown to co-localise with PCNA in replication foci.

Thus, various proteins interact with PCNA and it is thought that many ofthese interactions are mediated via a conserved PCNA interacting peptidesequence called the PIP-box (QxxL/I/MxxF/DF/Y [SEQ ID NO: 1205]),wherein x can be any amino acid. However, peptides that contain aPIP-box typically are extremely cytotoxic to human and animal cells andtherefore unsuitable for use in therapy.

However, APIM peptides have been shown to be useful in therapy.Specifically APIM peptides have been shown to be effective insensitizing cells to cytotoxic and cytostatic agents, particularlyDNA-damaging agents (WO 2009/104001) and indeed as an apoptosis-inducingcytotoxic agent in its own right (Müller et al., 2013. TargetingProliferating Cell Nuclear Antigen and Its Protein Interactions InducesApoptosis in Multiple Myeloma Cells, PLOS One, 8(7), e70430, pp. 1-12).Thus, APIM peptides have been shown to be useful in combination withcytotoxic and/or cytostatic agents in the treatment of a disorder orcondition where it is desirable to inhibit the growth of cells, or in atreatment which involves cytostatic therapy, i.e. to prevent or inhibitthe unwanted proliferation of cells.

In work leading up to the present invention, the inventors havesurprisingly determined that APIM peptides have a direct effect onmicroorganisms, i.e. APIM peptides have direct cytotoxic effects on avariety of microbial cells and also potentiate the effect of cytotoxicand/or cytostatic agents on microbial cells, i.e. sensitize microbialcells to various cytotoxic and/or cytostatic agents, particularly agentsthat act intracellularly, e.g. DNA-damaging agents, rather than agentsthat function at the cell membrane or cell wall, e.g. to permeabilizecells. Whilst not wishing to be bound by any specific theory, it ishypothesized that APIM peptides may interact with sliding clamp DNAproteins in microorganisms, thereby effecting DNA replication and repairin microbial cells, resulting in cytotoxicity and/or reduction in cellgrowth. In this respect, it is thought that microbial cells may containproteins that contain APIM motifs that are involved in modulatingvarious protein interactions that are critical for the numerous cellprocesses. Thus, the oligopeptidic compounds disclosed herein may beable to interfere with the interaction of said proteins with theirtarget binding partner(s) thereby inhibiting essential cellularfunctions, particularly DNA synthesis and repair, resulting in thestimulation of apoptosis or apoptosis-like cell death, or increasedsensitivity of the microbial cells to other cytotoxic and/or cytostaticagents.

Infectious diseases, also known as transmissible diseases orcommunicable diseases, comprise clinically evident illness, i.e.symptoms of disease, resulting from the infection, presence and growthof pathogenic biological agents, e.g. microbial organisms such asbacteria and fungi, in an individual host organism. In certain cases,infectious diseases may be asymptomatic for much or even all of theircourse in a given host. In the latter case, the disease may only bedefined as a “disease” (which by definition means an illness) in hostswho secondarily become ill after contact with an asymptomatic carrier.

Microbial infections do not always result in, or progress to, aclinically overt or symptomatic disease or disease state. For instance,a wound may become infected by one or more microbes, without resultingin an infectious disease. Furthermore, microbial growth in and on asubject may occur naturally, e.g. commensal growth, such asgastrointestinal microbial flora. Thus, a microbial infection may beviewed as any atypical, unwanted, undesirable, excessive and/or harmfulinfection and does not necessarily involve or result in a disease.

Transmission of a microbial pathogen can occur in various ways includingphysical contact, contaminated food, body fluids, objects, airborneinhalation, or through vector organisms. Infectious diseases aresometimes called “contagious” when they are easily transmitted bycontact with an ill person or their secretions. Thus, a contagiousdisease is a subset of infectious disease that is especially infectiveor easily transmitted.

Although only a relatively small proportion of microorganisms causedisease in otherwise healthy individuals, infectious diseases are one ofthe main contributors to global mortality and morbidity and a hugeamount of effort has gone into the discovery and development ofantimicrobial compounds, for both the treatment and prevention ofinfectious diseases.

However, ever since antimicrobials, particularly antibiotics, were firstused it was found that microbes, e.g. bacteria, could display intrinsicresistance to these drugs or could develop resistance to these drugs.Resistance of a microbe, e.g. bacterium or fungus, to an antibiotic orantimycotic can be viewed as a substantially greater tolerance, orreduced susceptibility, to the antibiotic or antimycotic compared to asensitive microbe or a typical or a wild type version of the microbe. Insome cases a microbe can be completely unaffected by exposure to anantibiotic or antimycotic. In this instance the microbe can beconsidered fully resistant to that antibiotic or antimycotic.

Multidrug resistance (MDR) in bacteria describes the situation where abacterium is resistant to at least three classes of drugs, specificallyin the context of bacteria, at least three classes of anti-microbial (ormore specifically anti-bacterial) agents. Antibiotics in one class arefunctionally unrelated, structurally unrelated, or both, to antibioticsin a different class. MDR in bacteria is thus often termed multipleanti-bacterial drug resistance or multiple antibiotic resistance. Theterms are used interchangeably in the art and herein. Bacteriadisplaying multidrug resistance phenotypes (or multipleantibacterial/antibiotic drug resistance phenotypes) are referred to asMDR bacteria (or sometimes MAR bacteria). Again, these terms are usedinterchangeably in the art and herein.

Antimicrobial, e.g. antibiotic/antimycotic, resistance mechanisms arenumerous. For instance, resistance may arise from cell impermeability,which physically prevents the antimicrobial from reaching its site ofaction in or on the cell; efflux mechanisms which prevent effectiveamounts of the antimicrobial reaching its site of action in or on themicrobe by rapidly removing the antimicrobial from the cell; metabolicmechanisms which breakdown the antimicrobial or convert theantimicrobial into a harmless (or less harmful) compound, or a compoundmore easily excreted; bypass mechanisms in which the microbe usesalternative pathways to those inhibited by the antimicrobial; or throughthe microbe having a form of the antimicrobial target (e.g. enzyme) thatis less sensitive to the antimicrobial or not having the target at all.

Development (or acquisition) of resistance can be through mutation. Forinstance, this may involve changes in the structure of the target of theantimicrobial that reduces the sensitivity of the target to theantimicrobial. It can also be a mutation in a pathway involved in theregulation of the cellular machinery involved in the metabolism orefflux of the antimicrobial. It can also be a mutation in theconstituents of the outer layers (e.g. the membranes/walls) of themicrobe that effects the permeability of the antimicrobial into themicrobe. In some instances multiple mutations must accumulate in orderfor a microbe to become resistant to a particular antimicrobial or classthereof.

Recent studies have indicated that the process of translesion synthesis(TLS) in bacteria contributes to the acquisition of antibioticresistance, e.g. in MDR strains. TLS is a cellular mechanism to tolerateDNA damage in which specific DNA polymerases (TLS polymerases) areexpressed that are capable of by-passing and leaving the DNA lesions inDNA for the possibility of removal later, thereby enabling the cell tocomplete the duplication of its genome. However, this damage tolerancemechanism is error-prone because TLS polymerases are commonlylow-fidelity enzymes and insert bases in a non-Watson Crick manneropposite the lesion and opposite undamaged DNA, and thus theirinaccurate synthesis introduces mutations. It is thought that thesemutations contribute to genetic diversity in bacteria and facilitate theacquisition of antibiotic resistance, particularly in MDR strains.

Many MDR species and strains of microbe exist today. For instance,bacterial genera from which MDR species and strains pose significantproblems for human and animal health include, but are not limited toPseudomonas, Acinetobacter, Burkholderia, Klebsiella, Providencia,Enterococcus and Staphylococcus.

Accordingly, there remains a need for effective therapies suitable forthe treatment of microbial infections, particularly diseases, disordersor conditions caused by, or associated with, microbial infections (e.g.infectious diseases caused by, associated with, or exacerbated by, amicrobial infection, e.g. a bacterial or fungal infection), which alsohave minimal side effects.

As mentioned above, the inventors have surprisingly found that APIMpeptides have a direct effect on microorganisms, i.e. APIM peptides havedirect cytotoxic effects on a variety of microbial cells and alsopotentiate the effect of cytotoxic and/or cytostatic agents on microbialcells. As discussed in more detail in the Examples, the inventors haveunexpectedly determined that oligopeptidic compounds comprising a PCNAinteracting motif (APIM motif) and an uptake peptide can be importedinto microbial cells (exemplified with yeast and both gram negative andgram positive bacteria), wherein the compounds have a cytotoxic and/orcytostatic effect, i.e. an anti-microbial effect, e.g. bactericidal(antibiotic) or fungicidal (antimycotic) effects. Thus, theoligopeptidic compounds described herein may find utility asanti-microbial agents alone and/or may enhance the effect of othercytotoxic and/or cytostatic agents, e.g. anti-bacterial or anti-fungalagents. Thus, for instance, the introduction of the agents describedherein, e.g. intravenously, may be useful in the treatment ofsepticaemia (an infection of the blood) or oral administration may beuseful in the treatment of, e.g. gastric ulcers caused by a bacterialinfection, such as Helicobacter pylori (a gram negative bacterium), orother infected wounds etc. Thus, it may be expected that the agentsdefined herein may be effective in the treatment of a number ofmicrobial infections including various infectious diseases, e.g.bacterial and/or fungal infections, or conditions caused or exacerbatedby, or associated with, an infectious disease.

These surprising findings have led the inventors to propose newtherapeutic uses for APIM peptides, i.e. peptides comprising a PCNAbinding motif, namely for use in treating an infectious disease orinfection or a disease or condition exacerbated or caused by aninfection, e.g. by acting directly on the infecting microbes and/oracting indirectly by potentiating the effects of other anti-microbialcompounds, particularly cytotoxic or cytostatic agents, e.g. DNAdamaging agents. Furthermore, the inventors have also demonstrated thatAPIM peptides may also potentiate the effects of DNA damaging radiation,particularly UV radiation.

As mentioned above, it is thought that the peptides of the invention actby interfering with DNA replication and repair mechanisms. This isdifferent to mechanism of action for many known anti-microbials, whichcommonly act by inhibiting cell wall synthesis, e.g. beta-lactams (suchas penicillins, cephalosporins, carbapenems, monobactams), polymyxins,or by inhibiting protein synthesis, e.g. microlides, aminoglycosides,tetracyclines etc. Whilst not wishing to be bound by theory it ishypothesised that APIM peptides may function by inferring with proteininteractions between the sliding clamp (e.g. the β-clamp in bacteria,such as E. coli) and DNA polymerases and other proteins involved in DNArepair, including TLS polymerases. This is particularly surprising inview of the differences between the conserved motif that mediates theinteractions between the β-clamp and polymerases in bacteria, QL[S/D]LF,and the APIM sequence. Thus, the antimicrobial agents of the presentinvention may be particularly useful in combating diseases or conditionscaused by MDR microbes, e.g. MDR bacteria, because the agents act on adifferent part of the cellular machinery to which resistance mechanismshave not yet evolved. Moreover, the antimicrobial agents of the presentinvention may interfere with the mechanisms associated with theacquisition of MDR.

The inventors have also determined that the peptides of the inventionare particularly effective at combating biofilms. In general terms abiofilm is a collection, or community, of microorganisms surrounded by amatrix of extracellular polymers (also known in the art as aglycocalyx). These extracellular polymers are typically polysaccharides,notably polysaccharides produced by the organisms themselves, but theycan contain other biopolymers as well. A biofilm will typically beattached to a surface, which may be inert or living, but it has alsobeen observed that biofilms may form from microorganisms attached toeach other or at any interface. Generally, therefore, a biofilm ischaracterised as a highly organised multicellular community ofmicroorganisms encased in, or surrounded by, an extracellular polymermatrix, generally a polysaccharide matrix, and typically in closeassociation with a surface or interface. Such a mode of growth isprotective to the microorganisms and renders them difficult to remove oreradicate (for example, as discussed further below, recalcitrant orresistant to anti-microbial agents or host defence or clearancemechanisms).

Biofilms cause significant commercial, industrial and medical problems,in terms of infections, contamination, fouling and spoilage etc, andthus the present invention provides a significant advantage in enablingor facilitating the combating of such biofilms, including both reducingor preventing their formation, and rendering them more susceptible toremoval or reduction, e.g. more susceptible to the effect ofanti-microbial agents (including disinfectants or antibiotics) or indeedin the case of an infection, to the immune response of the infectedhost. The efficacy of anti-microbial agents, both therapeutic andnon-therapeutic and including particularly antibiotics, may thus beenhanced.

Biofilms are found ubiquitously on a wide variety of surfaces orinterfaces (e.g. water/solid and water/gas (for example water/air)interfaces) if conditions conducive to microbial colonisation exist.Basically a biofilm will form wherever there are microorganisms and aninterface or surface, particularly a surface exposed to water ormoisture and biofilms are now recognised as the natural state ofmicrobial growth on such surfaces or interfaces.

The microorganisms in a biofilm community display properties at thecellular level (phenotype) that are not shared by their planktonic(free-floating) equivalents. In fact, it is believed that microorganismsin a biofilm are profoundly different from planktonic free-floatingcells. Further differences can also be observed at the community leveland are attributed to the effects of the extracellular matrix. Perhapsmost notable is the commonly observed phenomenon that microorganisms ina biofilm environment do not display the same susceptibilities toanti-microbial agents, e.g. antibiotics, anti-fungals and microbicides,and host immune defences or clearance mechanisms.

It is now becoming evident and increasingly documented that biofilms mayform in the case of microbial infections i.e. within or on an infectedhost. Thus biofilm formation may also occur on a “physiological” or“biological” surface, that is on an animate or biotic surface, or asurface on or in an infected host organism (e.g. a human or non-humananimal subject), for example on an internal or external body or tissuesurface. Such biofilm formation (or infection) on body tissues isincreasingly believed to contribute to various infective diseases,including for example native valve endocarditis (mitral, aortic,tricupsid, pulmonic heart valves), acute otitis media (middle ear),chronic bacterial prostatitis (prostate), cystic fibrosis (lungs),pneumonia (respiratory tract), periodontitis (tissues supporting theteeth, e.g. gingiva, periodontal ligament, alvelor bone).

Biofilm niches are also present when medical devices are implanted andthe formation of biofilm on such implanted (“in-dwelling”) devices canlead to clinical problems with infection at such sites, such asprosthetic valve endocarditis and device-related infection, for examplewith intrauterine devices, contact lenses, prostheses (e.g. prostheticjoints) and at catheterisation sites, for example with central venous orurinary catheters.

A significant problem and risk with such biofilm infections is thatmicroorganisms (or more particularly microcolonies) may break off ordetach from the biofilm, and enter other tissues, includingsignificantly the circulation. Such circulating biofilm-derivedmicroorganisms can cause further infections and lead to significantclinical problems, particularly as the detached circulatingmicroorganisms may have all the resistance characteristics of the parentcommunity.

Body or tissue surfaces which are dead or damaged (e.g. necrotic orinflamed) are particularly susceptible to biofilm infection. Wounds aresusceptible to infection and biofilm formation can occur in wounds thatdo not heal in a short amount of time. Wounds are an ideal environmentfor the formation of biofilms due to their susceptibility to bacterialcolonisation and the availability of substrate and surface for biofilmattachment. Problematically, infection of a wound often delays healingfurther and thus renders that wound more susceptible to biofilmformation and established infection. Wounds in which healing is delayed(so called chronic wounds) represent sites of particular concern withrespect to biofilm formation. However, evidence is increasingly growingthat both chronic and acute wounds may be sites of biofilm infection,with evidence of diverse microbial communities or populations in wounds,particularly chronic wounds, including anaerobic bacteria within chronicwounds.

Biofilm based infection is very difficult to treat and biofilmcontamination is very difficult to eradicate. Given the widespreadoccurrence of biofilms and the medical, environmental, industrial orother commercial problems they cause, any means of improving or enablingthe combating of biofilms would be very important, both clinically andcommercially. A need therefore exists for new methods of combatingbiofilms, both in clinical and industrial or commercial situations.

As noted above, it has been found that the peptides of the invention areeffective as anti-biofilm agents, e.g. capable of inhibiting orpreventing the formation of biofilms. Accordingly, the present inventionmay be seen also to provide new methods and means for combating biofilm,in vitro (e.g. on a product, material, device or implant), in vivo (e.g.at a wound site, including a surgical wound, a implant site etc.) or exvivo.

Thus, at is broadest, the invention can be seen to provide a method oftreating or preventing a microbial infection, more particularly amicrobial infectious disease or a disease or condition exacerbated orcaused by a microbial infection, particularly a bacterial or fungalinfectious disease or infection, said method comprising administering(particularly administering an effective amount of) an agent comprisingor encoding a peptide comprising an APIM motif or a composition (e.g. apharmaceutical composition) containing an agent comprising or encoding apeptide comprising an APIM motif to a subject in need thereof.

Thus, the invention provides an agent comprising or encoding a peptidecomprising an APIM motif or a composition (e.g. a pharmaceuticalcomposition) containing an agent comprising or encoding a peptidecomprising an APIM motif, for use in treating or preventing a microbialinfection, more particularly a microbial infectious disease or a diseaseor condition exacerbated or caused by a microbial infection,particularly a bacterial or fungal infectious disease or infection.

In another aspect, there is provided the use of an agent comprising orencoding a peptide comprising an APIM motif in the manufacture of amedicament for the treatment or prevention of a microbial infection,more particularly a microbial infectious disease or a disease orcondition exacerbated or caused by a microbial infection, particularly abacterial or fungal infectious disease or infection.

More particularly, the invention provides a method of treating orpreventing a microbial infection, more particularly a microbialinfectious disease or a disease or condition exacerbated or caused by amicrobial infection, particularly a bacterial or fungal infectiousdisease or infection, said method comprising administering (particularlyadministering an effective amount of) an agent or a composition (e.g. apharmaceutical composition) containing an agent to a subject in needthereof, wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and adomain that facilitates the cellular uptake of said compound,

wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) andwherein:

-   -   X₁ is a basic amino acid;    -   X₂ is an aromatic amino acid;    -   X₃ is an uncharged amino acid other than an aromatic amino acid,        Glycine (G) and Proline (P);    -   X₄ is any amino acid other than Proline (P), an acidic amino        acid or an aromatic amino acid;    -   X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding theoligopeptidic compound of (i).

In another aspect, there is provided an agent or a composition (e.g. apharmaceutical composition) containing an agent for use in treating orpreventing a microbial infection, more particularly a microbialinfectious disease or a disease or condition exacerbated or caused by amicrobial infection, particularly a bacterial or fungal infectiousdisease or infection, wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and adomain that facilitates the cellular uptake of said compound,

wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) andwherein:

-   -   X₁ is a basic amino acid;    -   X₂ is an aromatic amino acid;    -   X₃ is an uncharged amino acid other than an aromatic amino acid,        Glycine (G) and Proline (P);    -   X₄ is any amino acid other than Proline (P), an acidic amino        acid or an aromatic amino acid;    -   X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding theoligopeptidic compound of (i).

In an further aspect, there is provided the use of an agent in themanufacture of a medicament for the treatment or prevention of amicrobial infection, more particularly a microbial infectious disease ora disease or condition exacerbated or caused by a microbial infection,particularly a bacterial or fungal infectious disease or infection,wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and adomain that facilitates the cellular uptake of said compound,

wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) andwherein:

-   -   X₁ is a basic amino acid;    -   X₂ is an aromatic amino acid;    -   X₃ is an uncharged amino acid other than an aromatic amino acid,        Glycine (G) and Proline (P);    -   X₄ is any amino acid other than Proline (P), an acidic amino        acid or an aromatic amino acid;    -   X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding theoligopeptidic compound of (i).

In particular embodiments the invention provides a method of treating orpreventing a bacterial infection, said method comprising administeringan agent, or a composition containing an agent, to a subject in needthereof, wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and adomain that facilitates the cellular uptake of said compound,

wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) andwherein:

-   -   X₁ is a basic amino acid;    -   X₂ is an aromatic amino acid;    -   X₃ is an uncharged amino acid other than an aromatic amino acid,        Glycine (G) and Proline (P);    -   X₄ is any amino acid other than Proline (P), an acidic amino        acid or an aromatic amino acid; and    -   X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding theoligopeptidic compound of (i).

In a further embodiment, the invention provides an agent, or acomposition containing an agent, for use in treating or preventing abacterial infection in a subject, wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and adomain that facilitates the cellular uptake of said compound,

wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) andwherein:

-   -   X₁ is a basic amino acid;    -   X₂ is an aromatic amino acid;    -   X₃ is an uncharged amino acid other than an aromatic amino acid,        Glycine (G) and Proline (P);    -   X₄ is any amino acid other than Proline (P), an acidic amino        acid or an aromatic amino acid; and    -   X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding theoligopeptidic compound of (i).

In still further embodiments, the invention provides the use of an agentin the manufacture of a medicament for the treatment or prevention of abacterial infection in a subject, wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and adomain that facilitates the cellular uptake of said compound,

wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) andwherein:

-   -   X₁ is a basic amino acid;    -   X₂ is an aromatic amino acid;    -   X₃ is an uncharged amino acid other than an aromatic amino acid,        Glycine (G) and Proline (P);    -   X₄ is any amino acid other than Proline (P), an acidic amino        acid or an aromatic amino acid; and    -   X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding theoligopeptidic compound of (i).

As noted above, in some embodiments the agent may be used in combinationwith one or more additional active agents, e.g. a cytostatic orcytotoxic agent, in order to enhance the effect of that additionalactive agent, or to sensitise cells to the effect of said additionalactive agent, e.g. cytostatic or cytotoxic agent. However, in someembodiments, the agent as defined herein may be used alone, i.e. as theonly active agent capable of preventing or inhibiting microbial growth(e.g. having anti-microbial activity) in a composition and/ormedicament.

Accordingly, in yet another aspect, there is provided a method oftreating or preventing a microbial infection, more particularly amicrobial infectious disease or a disease or condition exacerbated orcaused by a microbial infection, particularly a bacterial or fungalinfectious disease or infection, said method comprising administering anagent or composition as defined herein, and separately, simultaneouslyor sequentially administering of one or more additional active agents,e.g. a cytostatic or cytotoxic agent, to a subject in need thereof.

Alternatively viewed, there is provided an agent or composition asdefined herein for use in combination with one or more additional activeagents, e.g. a cytostatic or cytotoxic agent, in the treatment orprevention of a microbial infection, more particularly a microbialinfectious disease or a disease or condition exacerbated or caused by amicrobial infection, particularly a bacterial or fungal infectiousdisease or infection.

Thus, there is provided the use of an agent as defined herein in themanufacture of a medicament for use in combination with one or moreadditional active agents, e.g. a cytostatic or cytotoxic agent, in thetreatment or prevention of a microbial infection, more particularly amicrobial infectious disease or a disease or condition exacerbated orcaused by a microbial infection, particularly a bacterial or fungalinfectious disease or infection.

In a particular embodiment, the invention provides a method of treatingor preventing a bacterial infection, said method comprisingadministering an agent or composition as defined herein, and separately,simultaneously or sequentially administering of one or more additionalactive agents, e.g. a cytostatic or cytotoxic agent, to a subject inneed thereof.

Alternatively viewed, there is provided an agent or composition asdefined herein for use in combination with one or more additional activeagents, e.g. an antibiotic, in the treatment or prevention of abacterial infection.

Thus, there is provided the use of an agent as defined herein in themanufacture of a medicament for use in combination with one or moreadditional active agents, e.g. an antibiotic, in the treatment orprevention of a bacterial infection.

Thus, in one embodiment the medicament may further comprise one or moreadditional active agents, such as a cytostatic or cytotoxic agent, e.g.an antibiotic.

The medicament may be in the form of a single composition comprisingboth the agent as defined herein and the one or more additional activeagents, e.g. cytostatic or cytotoxic agent, or it may be in the form ofa kit or product containing them for separate (e.g. simultaneous orsequential) administration.

There is thus also provided the use of an agent as defined herein in themanufacture of a medicament for the treatment or prevention of amicrobial infection, more particularly a microbial infectious disease ora disease or condition exacerbated or caused by a microbial infection,particularly a bacterial or fungal infectious disease or infection,wherein the medicament is administered separately, simultaneously orsequentially with one or more additional active agents, e.g. acytostatic or cytotoxic agent.

In another aspect, the invention provides a product containing an agentas defined herein together with one or more additional active agents,e.g. a cytostatic or cytotoxic agent, as a combined preparation forseparate, simultaneous or sequential use in the treatment or preventionof a microbial infection, more particularly a microbial infectiousdisease or a disease or condition exacerbated or caused by a microbialinfection, particularly a bacterial or fungal infectious disease orinfection.

In still further embodiments, there is provided the use of an agent asdefined herein in the manufacture of a medicament for the treatment orprevention of a bacterial infection, wherein the medicament isadministered separately, simultaneously or sequentially with one or moreadditional active agents, e.g. an antibiotic.

In another aspect, the invention provides a product containing an agentas defined herein together with one or more additional active agents,e.g. an antibiotic, as a combined preparation for separate, simultaneousor sequential use in the treatment or prevention of a bacterialinfection.

The agent as defined herein may be used to modulate or potentiate theeffect of one or more additional active agents, e.g. a cytostatic orcytotoxic agent. For instance, the agent may sensitize the microbialcell to the one or more additional active agents, e.g. a cytostatic orcytotoxic agent. Alternatively viewed, the one or more additional activeagents, such as a cytostatic or cytotoxic agent, may enhance, augment orimprove the anti-microbial effect of the agent defined herein. Thus, theagents defined herein may have the advantage of enabling lower doses ofanti-microbial agents to be effective and/or improving the efficacy ofanti-microbials against resistant strains.

In some embodiments the agent may be used in combination with DNAdamaging radiation, e.g. UV radiation, in order to enhance the effect ofthe radiation, or to sensitize cells to the effect of said radiation,e.g. UV radiation.

UV radiotherapy (also known as UV radiation therapy or UV lighttherapy), may be used in the treatment of various microbial infections,particularly bacterial infections. By “UV radiotherapy” is meant the useof UV radiation, preferably UVC radiation, i.e. radiation with awavelength of 200 nm to 290 nm.

Unfortunately UV radiotherapy is often unsuccessful at completelyeradicating microbial cells from a patient because it is often notpossible to deliver a sufficiently high dose of local radiation to killmicrobial cells without an unacceptably high risk of damage to thesurrounding normal tissue at the site of infection. It is also knownthat microbial cells show widely varying susceptibilities toradiation-induced cell death and ionizing radiation may only inhibitfurther cell growth, rather than eradicating the microbial cells assuch. Thus, there is a need to enhance the efficacy of radiotherapy bysensitizing cells to the effects of ionizing UV radiation.

Accordingly, the agents and compositions of the invention may be used toprovide such a sensitizing effect, in other words to enhance (oralternatively put to increase, augment, or potentiate) the effects of UVradiotherapy, particularly UVC radiotherapy, or to render microbialcells, e.g. bacterial cells, which may be present in an infection sitein a subject, more susceptible to the effects of said radiotherapy.Thus, they may find utility in any antimicrobial application whereradiotherapy is used. This may include any situation where it is desiredto kill, inhibit or eradicate bacterial cells, e.g. at an infection siteor in vitro.

The agents and compositions of the invention may thus be used as asensitizer of microbial cells, e.g. bacterial cells, to the effects ofUV radiation. By “sensitizer” is meant the use of the agents andcompositions of the invention to enhance the effect of UV radiation onmicrobial, e.g. bacterial, cells. This may be achieved by the inhibitionof the endogenous cellular DNA repair mechanisms, e.g. the TLS pathway.

Thus, the present invention encompasses an agent, or a compositioncontaining an agent, for use as a sensitizer for UV radiotherapy in thetreatment or prevention of a microbial, e.g. bacterial, infection in asubject, or in the treatment or prevention of a microbial, e.g.bacterial, infection in subject which involves UV radiotherapy (e.g. UVCradiotherapy), wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and adomain that facilitates the cellular uptake of said compound,

wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) andwherein:

-   -   X₁ is a basic amino acid;    -   X₂ is an aromatic amino acid;    -   X₃ is an uncharged amino acid other than an aromatic amino acid,        Glycine (G) and Proline (P);    -   X₄ is any amino acid other than Proline (P), an acidic amino        acid or an aromatic amino acid; and    -   X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding theoligopeptidic compound of (i).

In further embodiments, the methods of treatment defined above maycomprise UV radiotherapy (e.g. UVC radiotherapy), which may beadministered simultaneously, sequentially or separately to said agent orcomposition.

It will be evident that therapies involving the administration of UVradiation (UV radiotherapy) may be particularly useful for the treatmentof topical infections, e.g. infections of the skin or mucosal membranes,such as the oral cavity, oesophagus and/or eye. Thus, in someembodiments, the agent or composition for use as a sensitizer for UVradiation, or for use in methods comprising UV radiotherapy, may beformulated for topical administration, e.g. to the skin and/or muscosalmembrane. However, the invention is not limited to this aspect, as theagents and compositions of the invention may be combined with UVradiation to treat infections by other means, e.g. endoscopically or exvivo. For instance, a blood infection may be treated by administeringthe agent or composition of the invention to a subject and subsequentlyor contemporaneously irradiating the blood of said subject bycirculating the blood through an external tube exposed to UV radiation,i.e. akin to a dialysis machine.

The agents and compositions as defined herein thus have a therapeuticutility in any condition or clinical situation where it is desirable (orwhere it may be of benefit) to prevent or inhibit the growth ofmicrobial cells, e.g. bacterial and/or fungal cells.

A microbial infection includes any microbial infectious disease or adisease or condition exacerbated or caused by a microbial infection.However, as noted above, microbial infections do not always result in,or progress to, a disease or disease state. Thus, in some embodiments, amicrobial infection may be viewed specifically as a microbial infectionthat is not associated with a disease or condition. Hence, the abovetherapeutic uses and methods may be viewed as the treatment orprevention of an atypical, unwanted, undesirable, excessive and/orharmful microbial infection and/or a microbial infectious disease or adisease or condition exacerbated or caused by a microbial infection.

Thus, in yet another embodiment, the agent or composition as definedherein may be used as an anti-microbial, e.g. anti-bacterial oranti-fungal agent in in vitro or ex vivo methods, e.g. in methods ofcell culture or where the agent is used in the context of an abiotic orinanimate setting, e.g. to treat an inanimate surface to prevent,inhibit or reduce microbial colonisation and/or growth, e.g. fordecontamination, antiseptic or sterilisation purposes, or is applied toor contacted with a surface, material, substrate, product, device orsystem susceptible to microbial growth, e.g. contamination, such as inthe preparation of a medical device or implant.

Thus, the invention also provides an in vitro or ex vivo method ofadministering an agent or composition as defined herein to a microbialcell or cell culture, i.e. to inhibit or prevent the growth of one ormore microbes, e.g. bacteria or fungi. This may allow the identificationand/or characterisation of agents as defined, e.g. to determine the doseat which the agent is effective or determine which microbes areparticularly susceptible. Furthermore, the in vitro methods may beuseful to identify other anti-microbial compounds, e.g. compounds thatare weakly anti-microbial when used on their own, but which have usefulanti-microbial activity when used in combination with the agent of theinvention. Alternatively viewed, the invention provides the use of anagent or composition as defined herein in in vitro or ex vivo methods,e.g. microbial cell culture or in the context of an abiotic or inanimatesetting, e.g. to treat an inanimate surface (or product or material etc,e.g. as listed above) to prevent, inhibit or reduce microbialcolonisation and/or growth, e.g. for decontamination, antiseptic orsterilisation purposes, or for application or administration to asurface or system (etc, as above) susceptible to microbial growth, e.g.contamination.

Thus, in a particular embodiment, the invention provides an in vitromethod of:

(i) preventing, inhibiting or reducing bacterial colonisation and/orgrowth in or on a surface, product or material; or

(ii) preventing, inhibiting or reducing unwanted or undesirablebacterial colonisation and/or growth of a bacterial cell,

comprising administering an agent or composition as defined herein to asurface, product or material susceptible to microbial growth or amicrobial cell or a cell culture, optionally simultaneously,sequentially or separately administering one or more additional activeagents to said surface, product or material, cell or cell culture.

In some embodiments, the method further comprises a step of exposingsaid surface, product or material to UV radiation, prior to,contemporaneously with, or after administering said agent orcomposition.

As noted above, the agent or composition as defined herein is used toprevent a microbial infection or contamination, e.g. in circumstanceswhere there is an increased probability of an infection, such as insurgery or in the treatment of a wound. Thus, in some embodiments theagent or composition as defined herein may be provided or administeredvia a product, device, implant or material to which the agent orcomposition has been applied, impregnated or chemically bonded. In thisrespect, the oligopeptidic compounds defined herein are commonlypositively charged and such compounds will readily adhere to varioussurfaces without the need for additional adhesives. However, the use ofadhesives or other methods of bonding the agents of the invention toproducts, devices, implants or materials is contemplated herein.

Hence, a further aspect of the invention comprises the provision of aproduct, material, device or implant which is coated, impregnated orchemically bonded with an agent or composition as described herein. Theinvention also extends to the use of such products, materials, devicesor implants in the methods and uses as described herein.

Thus, yet another aspect of the invention comprises a method ofproducing product, material, device or implant (e.g. an aseptic product,material, device or implant) which is coated, impregnated or chemicallybonded with an agent or composition as defined herein, comprisingproviding a product, material, device or implant and coating orimpregnating said device with said agent or composition, or chemicallybonding said agent or composition to said product, material, device orimplant. Alternatively viewed, the invention may be seen to provide theuse of an agent or composition as defined herein in the production of aproduct, material, device or implant, particularly in the production ofan aseptic product, material, device or implant.

As discussed above, the agents and compositions of the invention mayalso be used to combat biofilms, including both on biotic and abioticsurfaces. Thus, the methods and uses discussed above may be for use incombating biofilm infection or to combat biofilm formation on inanimatesurfaces e.g. for disinfection and cleaning purposes. Alternativelyviewed, the bacterial infection or bacterial colonisation and/or growthmay be a biofilm.

Thus, in some embodiments, the invention may provide a method ofpreventing or inhibiting the formation of a bacterial biofilm on aproduct, material, device or implant, said method comprising:

(i) providing a product, material, device or implant; and

(ii) coating or impregnating said device with said agent or composition,or chemically bonding said agent or composition to said product,material, device or implant.

Thus, an oligopeptidic compound (e.g. a peptide) capable of interactingwith PCNA may contain or comprise a peptide motif (or sequence) that maybe defined generally as:

X₁X₂X₃X₄X₅ (SEQ ID NO: 1), wherein:

-   -   X₁ is a basic amino acid;    -   X₂ is an aromatic amino acid;    -   X₃ is an uncharged amino acid other than an aromatic amino acid,        Glycine (G) and Proline (P);    -   X₄ is any amino acid other than Proline (P), an acidic amino        acid or an aromatic amino acid;    -   X₅ is a basic amino acid or Proline (P).

In order that the oligopeptidic compound, which is capable ofinteracting with PCNA, or its encoding nucleic acid, may function in themethods and uses of the invention, the compound must be capable ofentering the cell, i.e. crossing the cell membrane and cell wall, ifpresent, into the cytosol (cytoplasm), and optionally into one or moreother cellular locations, e.g. the nucleus. Whilst this may be achievedusing any convenient mechanism, such as with a liposome, as noted above,the inventors have surprisingly and advantageously found that uptake ofthe oligopeptidic compound may be achieved by associating theoligopeptidic compound with one or more molecules that are known to becapable of facilitating the uptake of molecules into animal cells, e.g.an import peptide.

Thus, the inventors have determined that is it particularly advantageousto generate an oligopeptidic compound that comprises a domain thatassists the transit of the compound across the cell membrane, i.e. togenerate a fusion peptide or chimeric peptide (a peptide formed from twoor more domains that are not normally found together in nature). Forinstance, a peptide comprising a cell membrane permeable motif, e.g. acell penetrating peptide (an uptake or import peptide, or a peptidetransduction domain). In some embodiments (e.g. wherein theoligopeptidic compound is for use as an antimycotic), the fusion peptide(an oligopeptidic compound) may optionally comprise further sequences tofacilitate the targeting of the peptide (i.e. to direct the peptide) toa particular sub-cellular location, e.g. a target peptide, signalpeptide or transit peptide. Whilst not wishing to be bound by theory, itis thought that it is the specific combination of a domain capable ofinteracting with PCNA and a domain that facilitates the uptake of theoligopeptidic compound, that results in the antimicrobial properties ofthe oligopeptidic compounds disclosed herein.

As the oligopeptidic compound comprises a PCNA interacting motif and adomain that facilitates its uptake, it will be evident that the compoundcomprises at least 5 residues and the final size of the compound will bedependent on the size and number of the domains that make up saidcompound, i.e. the PCNA interacting motif and uptake (import) peptidemay be viewed as domains of the oligopeptidic compound. Thus, a domainmay be viewed as a distinct portion (i.e. a sequence within thefull-length peptidic sequence) of the oligopeptidic compound that can beassigned or ascribed a particular function or property.

In some embodiments, the oligopeptidic compound for use in the methodsand uses of the invention comprises at least 2 domains, i.e. the PCNAinteracting motif domain and the domain that facilitates the cellularuptake of said compound, e.g. uptake (import) peptide sequence domain.However, the oligopeptidic compound may comprise additional domains thatmay facilitate its function and/or stability, e.g. the capacity of thepeptide to interact with its target, i.e. PCNA or an equivalent protein,such as the β-clamp protein from bacteria, e.g. E. coli. Thus, theoligopeptidic compound may comprise at least 2, 3, 4 or 5 domains, e.g.6, 7, 8, 9, 10, 12, 15 or more domains. For example, in some embodimentsthe oligopeptidic compound may comprise one or more linker domains, i.e.a domain that interspaces between two other domains, i.e. occupies thespace in between and connects two domains of the oligopeptidic compound.In further embodiments, the oligopeptidic compound may comprise a domainthat directs the oligopeptidic compound to a cellular or subcellularlocation, e.g. a signal peptide (also known as a target or transitpeptide), such as a nuclear localization signal (NLS) sequence. In stillfurther embodiments, the one or more linker domains may have anadditional function, i.e. a linker domain may also function as a signalpeptide, e.g. a NLS. Alternatively put, a signal peptide domain mayfunction as a linker domain in some embodiments.

In an exemplary embodiment, the oligopeptidic compound may comprise aPCNA interacting motif domain, a domain that facilitates its cellularuptake (e.g. an uptake (import) peptide sequence domain) and a linkerdomain. In a further exemplary embodiment, the oligopeptidic compoundmay also comprise a nuclear localisation signal sequence domain. Instill another embodiment the nuclear localization signal sequence domainmay function as a linker domain.

Thus, it will be seen that in such embodiments the agent of theinvention may take the form of a construct containing (i.e. comprising)an oligopeptidic compound which comprises a PCNA interacting motif asdefined herein, together with a domain that facilitates its cellularuptake (e.g. an uptake peptide sequence) and optionally additionaldomains. In this aspect the invention may accordingly be seen to providea construct comprising an oligopeptidic compound which is capable ofinteracting with PCNA.

As noted above the PCNA motif of the invention has been determined tomediate the interaction of an oligopeptidic compound (e.g. peptide) orprotein containing such a motif with PCNA. However, the inventors haveunexpectedly determined that the oligopeptidic compounds may alsointeract with PCNA equivalent proteins, e.g. proteins that arefunctionally equivalent and/or structurally similar to PCNA, such as theβ-clamp protein from bacteria, e.g. E. coli. Thus, it is thought thatthe oligopeptidic compounds may interact with proteins in microbialcells that are functionally equivalent to PCNA, but structurallydistinct. In some instances, the oligopeptidic compounds may interactwith proteins in microbial cells that are structurally similar to PCNA,but functionally distinct. However, whilst the oligopeptidic compound ofthe invention may not function using the same mechanism in each type ofcell, i.e. eukaryotic and prokaryotic, the compounds of the inventionmay be characterised insofar as they must be capable of interacting withPCNA, i.e. the oligopeptidic compounds for use in the methods and usesof the invention must be competent and/or proficient PCNA interactingmolecules. The PCNA protein used to determine the capacity and/oraffinity of the oligopeptidic compound:PCNA interaction may be from anysuitable source, e.g. a PCNA from any animal, particularly a mammal suchas a human, rodent (e.g. mouse, rat) or any other non-human animal. Inpreferred embodiments, the oligopeptidic compound:PCNA interaction isdetermined, characterised or assessed using human PCNA protein.

The interaction may be direct or indirect, and may involve directbinding of the motif to the PCNA protein, or the motif may bindindirectly, for example binding may be mediated by another molecule.This reference to “PCNA-interacting” or “PCNA-binding” can thus includeany form of interaction, and both direct and indirect binding.

Any reference herein to a “motif” should be understood to meanX₁X₂X₃X₄X₅ as defined herein.

X₁ is preferably selected from lysine (K), arginine (R), histidine (H),ornithine (Orn), methyllysine (MeK), diaminobutyric acid (Dbu),citrulline (Cit), acetyllysine (AcK), and any suitable basic amino acidselected from the non-conventional amino acids in Table 2. Whilst thestandard or conventional basic amino acids are preferred, e.g. K, R andH, particularly K and R, these may be substituted by any functionallyequivalent non-conventional basic amino acid.

X₂ is preferably selected from phenylalanine (F), tryptophan (W),tyrosine (Y), tert.-butylglycine, cyclohexylalanine,tert.-butylphenylalanine, biphenylalanine and tri tert.-butyltryptophan(in certain embodiments this list may exclude F). Whilst the standard orconventional aromatic amino acids are preferred, e.g. F, W and Y, thesemay be substituted by any functionally equivalent non-conventionalaromatic amino acid, e.g. from Table 2. In some embodiments, X₂ may beselected from W and Y, F and Y, or F and W or in specific embodiments X₂may be F or W or Y, or a functionally equivalent non-conventionalaromatic amino acid.

The binding of the motif to PCNA may in certain embodiments be improvedwhen X₂ is W or Y. Thus, in one embodiment, X₂ is not F. However, asindicated above, in other embodiments it may be F.

X₃ is preferably a hydrophobic or polar amino acid, particularly analiphatic amino acid or polar amino acid. Thus, in some embodiments, X₃may be selected from leucine (L), isoleucine (I), valine (V), alanine(A) methionine (M), norleucine (Nor), serine (S), threonine (T),glutamine (Q), aspargine (N) or cysteine (C) or any suitable hydrophobicor polar amino acid selected from the non-conventional amino acids inTable 2. More particularly, X₃ may be selected from L, I, V, A, M, Nor,S or T and any suitable hydrophobic (preferably aliphatic) or polar(preferably a polar amino acid that does not contain an amine group(NH₂) in the R-group) amino acid selected from the non-conventionalamino acids in Table 2. Preferably, X₃ is not N or Q or anon-conventional functional equivalent thereof and/or in certainembodiments X₃ is not M, S and/or T or a non-conventional functionalequivalent thereof. X₃ may not be glycine (G) or proline (P) and thislimitation is also intended to exclude non-conventional functionalequivalents thereof.

Thus, in some embodiments, X₃ may be selected from L, I, A, V, M, S andT, and preferably from L, I, A, V, S and T and optionallynon-conventional functional equivalents thereof.

In some embodiments, X₃ may be a hydrophobic, and more preferably analiphatic amino acid. Thus, in some embodiments, X₃ may be selected fromL, I, A, V, M, and preferably from L, I, V and A and optionallynon-conventional functional equivalents thereof.

X₄ is preferably a hydrophobic, polar, basic or thiol-containing aminoacid. Thus, in some embodiments X₄ an aliphatic amino acid or a polaramino acid. In some preferred embodiments, the polar amino acid does notcontain an amine group (NH₂) in the R-group. Thus, X₄ preferably may beselected from L, I, V, A, M, Nor, S, T, Q, N, H, K, R, G or C and anysuitable hydrophobic (preferably aliphatic) or polar (preferably a polaramino acid that does not contain an amine group (NH₂) in the R-group),basic or thiol-containing amino acid selected from the non-conventionalamino acids in Table 2. Thus, in some embodiments, the basic amino acidmay be selected from the amino acids as defined in X₁. In certainembodiments X₄ is not C or a non-conventional functional equivalentthereof and/or N or Q or a non-conventional functional equivalentthereof. In certain embodiments X₄ is not H and preferably X₄ is not R,K or H or a non-conventional functional equivalent thereof. In stillfurther embodiments X₄ is not S or T or a non-conventional functionalequivalent thereof. X₄ may not be P, an aromatic amino acid (as definedin X₂) or acidic amino acid, e.g. aspartic acid (D) or glutamic acid(E), and this limitation is also intended to exclude non-conventionalfunctional equivalents thereof.

Thus, in some embodiments, X₄ may be selected from L, V, I, A, M, S, Tand G, and preferably from L, V, A, I, S and T and optionallynon-conventional functional equivalents thereof.

In other embodiments, X₄ may be a hydrophobic amino acid, and morepreferably an aliphatic amino acid or G. Thus, in some embodiments, X₄may be selected from L, I, A, V, M, and G and preferably from L, V, Iand A and optionally non-conventional functional equivalents thereof.

X₅ is preferably selected from K, R, H, Orn, MeK, Dbu, Cit, AcK, P andany functionally equivalent amino acid selected from thenon-conventional amino acids in Table 2. Whilst the standard orconventional amino acids are preferred, e.g. K, R, H and P, particularlyK, R and H, e.g. K and R, these may be substituted by any functionallyequivalent non-conventional basic amino acid.

Thus, in some embodiments X₅ is a basic amino acid, preferably selectedfrom K, R and H and optionally non-conventional functional equivalentsthereof.

Thus, in some embodiments X₃ and/or X₄ is a polar amino acid.Accordingly, in certain embodiments only one of X₃ and X₄ is a polaramino acid.

In some embodiments X₄ and/or X₅ is a basic amino acid. Accordingly, incertain embodiments X₅ is a basic amino acid.

A functionally equivalent amino acid may be defined as an amino acidthat may be used as a substitute in a peptide or protein for aconventional amino acid without affecting significantly the function ofthe peptide or protein (or an amino acid that would not be expected toaffect or alter significantly the function of the peptide or protein),e.g. an amino acid that has similar structural and/or chemicalproperties to the conventional amino acid. Thus, a functionallyequivalent amino acid may be viewed as having the base structure of astandard amino acid, with a non-standard or non-conventional R-groupthat is structurally and/or chemically similar to the standard R-group.Preferably, the R-group is structurally similar to the standard R-groupof the amino acid being substituted.

A conventional or standard amino acid is an amino acid that is used invivo to produce a polypeptide or protein molecule, i.e. a proteinogenicamino acid. In other words, an amino acid with a standard orconventional R-group or an amino acid which possesses a side chain thatis coded for by the standard genetic code, i.e. “coded amino acids”.

Thus, the invention may provide an oligopeptidic compound comprising themotif[R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T/N/Q/C]-[L/I/V/A/M/G/S/T/N/Q/R/H/K/C]-[K/R/H/P](SEQ ID NO: 2), wherein said oligopeptidic compound is capable ofinteracting with PCNA.

In another embodiment the motif may be defined as:

(SEQ ID NO: 3) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T/N/Q]-[L/I/V/A/M/G/S/T/N/Q/R/H/K]-[K/R/H/P]

In another embodiment the motif may be defined as:

(SEQ ID NO: 4) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G/S/T/N/Q/R/H/K]-[K/R/H/P]

In another embodiment the motif may be defined as:

(SEQ ID NO: 5) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G/S/T/N/Q/R/H/K]-[K/R/H].

In another embodiment the motif may be defined as:

(SEQ ID NO: 6) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G/S/T/R/K]-[K/R/H].

In another embodiment the motif may be defined as:

(SEQ ID NO: 7) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G/S/T]-[K/R/H].

In another embodiment the motif may be defined as:

(SEQ ID NO: 8) [R/K]-[W/F/Y]-[L/I/V/A/M/S/T]- [L/I/V/A/M/G/S/T]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 9) [R/K]-[W/F]-[L/I/V/A/M/S/T]- [L/I/V/A/M/G/S/T]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 10) [R/K]-[W/F]-[L/I/V/A/M/T]- [L/I/V/A/M/G/S/T]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 11) [R/K]-[W/F]-[L/I/V/A/M/T]-[L/I/V/A/M/S/T]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 12) [R/K]-[W/F]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 13) [R/K]-[W/F]-[L/I/A/V/M/T]-[L/I/V/A/M/S/T]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 14) [R/K]-[W/F]-[L/I/V/A/M/S/T]-[L/V/A/I/S/T]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 15) [R/K]-[W/F]-[L/V/I/A/T]-[L/V/A/I/S/T]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 16) [R]-[W/F/Y]-[L/V/I/A]-[L/V/A/S/T/M]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 17) [R]-[W/F/Y]-[L/V/I/A/T]-[L/V/A/S/T/M]-[K].

In another embodiment the motif may be defined as:

(SEQ ID NO: 18) [R/K]-[F/Y]-[L/V/I/A]-[L/V/A/I/M]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 19) [R/K]-[F/W/Y]-[L/I/V/A]-[L/I/V/A]-[K/R].

In yet another embodiment the motif may be defined as:

(SEQ ID NO: 20) [R/K]-[W/Y]-[L/V/I/A/S/T]-[L/V/A/S/T/M]-[K/R].

In yet another embodiment the motif may be defined as:

(SEQ ID NO: 21) [K]-[F/Y/W]-[L/V/I/A/T]-[L/V/A/I/S/T/M]-[K].

In some embodiments X₁ and X₂ are RW, RF, KF, KW, RY or KY.

In some embodiments X₃ and X₄ are LL, LA, LV, AL, VL, VI, LI, IL, VV,VA, IV, II, AV, IA, AI, AM, LM, LS, LT, IS, MV, TV, AA, IM, LN, LQ, VM,TL, SL, IT, VT, LG, MA, ML, NL, QL, QI, TI, SI, AS, VS, SV, CA, IG, LR,VR, TK or IR. In some embodiments X₃ and X₄ are LL, LA, LV, AL, VL, VI,LI, IL, VV, VA, IV, II, AV, IA, AI, AM, LM, LS, LT, IS, MV, TV, AA, IM,LN, LQ or VM. In some embodiments X₃ and X₄ are LV, IV, SV, LS, AV, LG,LA, IR, LR, VR, AR, IK, LK, VK or AK. In particularly preferredembodiments X₃ and X₄ are LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV,II, AV, IA, AI, AM, LM, LS or LT, preferably LL, LA, LV, AL, VL, VI, LI,IL, VV, VA, IV, II, AV, IA or AI. Thus, in certain embodiments X₃ and X₄are not any one or more of AG, AC, CC, NN, QQ, NQ, QN, TS, SS, ST or TT.In yet further embodiments X₃ and X₄ are SL, LS, SV, LT or AV.

In some embodiments X₂ and X₃ are not FS or FT. In some embodiments X₂and X₃ are FS or FT.

In some embodiments X₅ is K. In some embodiments X₅ is P.

Thus, in a preferred embodiment, the oligopeptidic compound has orcomprises the sequence RWLVK (SEQ ID NO: 28). In other preferredembodiments, the oligopeptidic compound has or comprises a sequenceselected from any one or more of:

(SEQ ID NO: 22) RWLLK; (SEQ ID NO: 23) RFLLK; (SEQ ID NO: 24) RYLLK;(SEQ ID NO: 25) RWLLR; (SEQ ID NO: 26) RFLLR; (SEQ ID NO: 27) RYLLR;(SEQ ID NO: 28) RWLVK; (SEQ ID NO: 29) RFLVK; (SEQ ID NO: 30) RYLVK;(SEQ ID NO: 31) RWLVR; (SEQ ID NO: 32) RFLVR; (SEQ ID NO: 33) RYLVR;(SEQ ID NO: 34) RWIVK; (SEQ ID NO: 35) RFIVK; (SEQ ID NO: 36) RYIVK;(SEQ ID NO: 37) RWIVR; (SEQ ID NO: 38) RFIVR; (SEQ ID NO: 39) RYIVR;(SEQ ID NO: 40) RWLSK; (SEQ ID NO: 41) RFLSK; (SEQ ID NO: 42) RYLSK;(SEQ ID NO: 43) RWLSR; (SEQ ID NO: 44) RFLSR; (SEQ ID NO: 45) RYLSR;(SEQ ID NO: 46) RWISK; (SEQ ID NO: 47) RFISK; (SEQ ID NO: 48) RYISK;(SEQ ID NO: 49) RWISR; (SEQ ID NO: 50) RFISR; (SEQ ID NO: 51) RYISR;(SEQ ID NO: 52) RWSVK; (SEQ ID NO: 53) RFSVK; (SEQ ID NO: 54) RYSVK;(SEQ ID NO: 55) RWSVR; (SEQ ID NO: 56) RFSVR; (SEQ ID NO: 57) RYSVR;(SEQ ID NO: 58) RWAVK; (SEQ ID NO: 59) RFAVK; (SEQ ID NO: 60) RYAVK;(SEQ ID NO: 61) RWAVR; (SEQ ID NO: 62) RFAVR; (SEQ ID NO: 63) RYAVR;(SEQ ID NO: 64) RWLGR; (SEQ ID NO: 65) RFLGR; (SEQ ID NO: 66) RYLGR;(SEQ ID NO: 67) RWLGK; (SEQ ID NO: 68) RFLGK; (SEQ ID NO: 69) RYLGK;(SEQ ID NO: 70) RWLAR; (SEQ ID NO: 71) RFLAR; (SEQ ID NO: 72) RYLAR;(SEQ ID NO: 73) RWLAK; (SEQ ID NO: 74) RFLAK; (SEQ ID NO: 75) RYLAK;(SEQ ID NO: 76) RWLTK; (SEQ ID NO: 77) RFLTK; (SEQ ID NO: 78) RYLTK;(SEQ ID NO: 79) RWLTR; (SEQ ID NO: 80) RFLTR; (SEQ ID NO: 81) RYLTR;(SEQ ID NO: 82) RWITK; (SEQ ID NO: 83) RFITK; (SEQ ID NO: 84) RYITK;(SEQ ID NO: 85) RWITR; (SEQ ID NO: 86) RFITR; (SEQ ID NO: 87) RYITR;(SEQ ID NO: 88) RWTVK; (SEQ ID NO: 89) RFTVK; (SEQ ID NO: 90) RYTVK;(SEQ ID NO: 91) RWTVR; (SEQ ID NO: 92) RFTVR; (SEQ ID NO: 93) RYTVR;(SEQ ID NO: 94) RWIRK; (SEQ ID NO: 95) RFIRK; (SEQ ID NO: 96) RYIRK;(SEQ ID NO: 97) RWIRR; (SEQ ID NO: 98) RFIRR; (SEQ ID NO: 99) RYIRR;(SEQ ID NO: 100) RWLRK; (SEQ ID NO: 101) RFLRK; (SEQ ID NO: 102) RYLRK;(SEQ ID NO: 103) RWLRR; (SEQ ID NO: 104) RFLRR; (SEQ ID NO: 105) RYLRR;(SEQ ID NO: 106) KWLLK; (SEQ ID NO: 107) KFLLK; (SEQ ID NO: 108) KYLLK;(SEQ ID NO: 109) KWLLR; (SEQ ID NO: 110) KFLLR; (SEQ ID NO: 111) KYLLR;(SEQ ID NO: 112) KWLVK; (SEQ ID NO: 113) KFLVK; (SEQ ID NO: 114) KYLVK;(SEQ ID NO: 115) KWLVR; (SEQ ID NO: 116) KFLVR; (SEQ ID NO: 117) KYLVR;(SEQ ID NO: 118) KWIVK; (SEQ ID NO: 119) KFIVK; (SEQ ID NO: 120) KYIVK;(SEQ ID NO: 121) KWIVR; (SEQ ID NO: 122) KFIVR; (SEQ ID NO: 123) KYIVR;(SEQ ID NO: 124) KWLSK; (SEQ ID NO: 125) KFLSK; (SEQ ID NO: 126) KYLSK;(SEQ ID NO: 127) KWLSR; (SEQ ID NO: 128) KFLSR; (SEQ ID NO: 129) KYLSR;(SEQ ID NO: 130) KWISK; (SEQ ID NO: 131) KFISK; (SEQ ID NO: 132) KYISK;(SEQ ID NO: 133) KWISR; (SEQ ID NO: 134) KFISR; (SEQ ID NO: 135) KYISR;(SEQ ID NO: 136) KWSVK; (SEQ ID NO: 137) KFSVK; (SEQ ID NO: 138) KYSVK;(SEQ ID NO: 139) KWSVR; (SEQ ID NO: 140) KFSVR; (SEQ ID NO: 141) KYSVR;(SEQ ID NO: 142) KWAVK; (SEQ ID NO: 143) KFAVK; (SEQ ID NO: 144) KYAVK;(SEQ ID NO: 145) KWAVR; (SEQ ID NO: 146) KFAVR; (SEQ ID NO: 147) KYAVR;(SEQ ID NO: 148) KWLGR; (SEQ ID NO: 149) KFLGR; (SEQ ID NO: 150) KYLGR;(SEQ ID NO: 151) KWLGK; (SEQ ID NO: 152) KFLGK; (SEQ ID NO: 153) KYLGK;(SEQ ID NO: 154) KWLAR; (SEQ ID NO: 155) KFLAR; (SEQ ID NO: 156) KYLAR;(SEQ ID NO: 157) KWLAK; (SEQ ID NO: 158) KFLAK; (SEQ ID NO: 159) KYLAK;(SEQ ID NO: 160) KWLTK; (SEQ ID NO: 161) KFLTK; (SEQ ID NO: 162) KYLTK;(SEQ ID NO: 163) KWLTR; (SEQ ID NO: 164) KFLTR; (SEQ ID NO: 165) KYLTR;(SEQ ID NO: 166) KWITK; (SEQ ID NO: 167) KFITK; (SEQ ID NO: 168) KYITK;(SEQ ID NO: 169) KWITR; (SEQ ID NO: 170) KFITR; (SEQ ID NO: 171) KYITR;(SEQ ID NO: 172) KWTVK; (SEQ ID NO: 173) KFTVK; (SEQ ID NO: 174) KYTVK;(SEQ ID NO: 175) KWTVR; (SEQ ID NO: 176) KFTVR; (SEQ ID NO: 177) KYTVR;(SEQ ID NO: 178) KWLRK; (SEQ ID NO: 179) KFLRK; (SEQ ID NO: 180) KYLRK;(SEQ ID NO: 181) KWLRR; (SEQ ID NO: 182) KFLRR; (SEQ ID NO: 183) KYLRR;(SEQ ID NO: 184) KWIRK; (SEQ ID NO: 185) KFIRK; (SEQ ID NO: 186) KYIRK;(SEQ ID NO: 187) KWIRR; (SEQ ID NO: 188) KFIRR; (SEQ ID NO: 189) KYIRR;(SEQ ID NO: 190) RWVVK; (SEQ ID NO: 191) RFVVK; (SEQ ID NO: 192) RYVVK;(SEQ ID NO: 193) RWVVR; (SEQ ID NO: 194) RFVVR; (SEQ ID NO: 195) RYVVR;(SEQ ID NO: 196) KWVVK; (SEQ ID NO: 197) KFVVK; (SEQ ID NO: 198) KYVVK;(SEQ ID NO: 199) KWVVR; (SEQ ID NO: 200) KFVVR; (SEQ ID NO: 201) KYVVR;(SEQ ID NO: 202) RWALK; (SEQ ID NO: 203) RFALK; (SEQ ID NO: 204) RYALK;(SEQ ID NO: 205) RWALR; (SEQ ID NO: 206) RFALR; (SEQ ID NO: 207) RYALR;(SEQ ID NO: 208) KWALK; (SEQ ID NO: 209) KFALK; (SEQ ID NO: 210) KYALK;(SEQ ID NO: 211) KWALR; (SEQ ID NO: 212) KFALR; (SEQ ID NO: 213) KYALR;(SEQ ID NO: 214) RWVLK; (SEQ ID NO: 215) RFVLK; (SEQ ID NO: 216) RYVLK;(SEQ ID NO: 217) RWVLR; (SEQ ID NO: 218) RFVLR; (SEQ ID NO: 219) RYVLR;(SEQ ID NO: 220) KWVLK; (SEQ ID NO: 221) KFVLK; (SEQ ID NO: 222) KYVLK;(SEQ ID NO: 223) KWVLR; (SEQ ID NO: 224) KFVLR; (SEQ ID NO: 225) KYVLR;(SEQ ID NO: 226) RWILK; (SEQ ID NO: 227) RFILK; (SEQ ID NO: 228) RYILK;(SEQ ID NO: 229) RWILR; (SEQ ID NO: 230) RFILR; (SEQ ID NO: 231) RYILR;(SEQ ID NO: 232) KWILK; (SEQ ID NO: 233) KFILK; (SEQ ID NO: 234) KYILK;(SEQ ID NO: 235) KWILR; (SEQ ID NO: 236) KFILR; (SEQ ID NO: 237) KYILR;(SEQ ID NO: 238) RWVIK; (SEQ ID NO: 239) RFVIK; (SEQ ID NO: 240) RYVIK;(SEQ ID NO: 241) RWVIR; (SEQ ID NO: 242) RFVIR; (SEQ ID NO: 243) RYVIR;(SEQ ID NO: 244) KWVIK; (SEQ ID NO: 245) KFVIK; (SEQ ID NO: 246) KYVIK;(SEQ ID NO: 247) KWVIR; (SEQ ID NO: 248) KFVIR; (SEQ ID NO: 249) KYVIR;(SEQ ID NO: 250) RWIIK; (SEQ ID NO: 251) RFIIK; (SEQ ID NO: 252) RYIIK;(SEQ ID NO: 253) RWIIR; (SEQ ID NO: 254) RFIIR; (SEQ ID NO: 255) RYIIR;(SEQ ID NO: 256) KWIIK; (SEQ ID NO: 257) KFIIK; (SEQ ID NO: 258) KYIIK;(SEQ ID NO: 259) KWIIR; (SEQ ID NO: 260) KFIIR; (SEQ ID NO: 261) KYIIR;(SEQ ID NO: 262) RWLIK; (SEQ ID NO: 263) RFLIK; (SEQ ID NO: 264) RYLIK;(SEQ ID NO: 265) RWLIR; (SEQ ID NO: 266) RFLIR; (SEQ ID NO: 267) RYLIR;(SEQ ID NO: 268) KWLIK; (SEQ ID NO: 269) KFLIK; (SEQ ID NO: 270) KYLIK;(SEQ ID NO: 271) KWLIR; (SEQ ID NO: 272) KFLIR; (SEQ ID NO: 273) KYLIR;(SEQ ID NO: 274) RWIAK; (SEQ ID NO: 275) RFIAK; (SEQ ID NO: 276) RYIAK;(SEQ ID NO: 277) RWIAR; (SEQ ID NO: 278) RFIAR; (SEQ ID NO: 279) RYIAR;(SEQ ID NO: 280) KWIAK; (SEQ ID NO: 281) KFIAK; (SEQ ID NO: 282) KYIAK;(SEQ ID NO: 283) KWIAR; (SEQ ID NO: 284) KFIAR; (SEQ ID NO: 285) KYIAR;(SEQ ID NO: 286) RWVAK; (SEQ ID NO: 287) RFVAK; (SEQ ID NO: 288) RYVAK;(SEQ ID NO: 289) RWVAR; (SEQ ID NO: 290) RFVAR; (SEQ ID NO: 291) RYVAR;(SEQ ID NO: 292) KWVAK; (SEQ ID NO: 293) KFVAK; (SEQ ID NO: 294) KYVAK;(SEQ ID NO: 295) KWVAR; (SEQ ID NO: 296) KFVAR; (SEQ ID NO: 297) KYVAR;(SEQ ID NO: 1206) RFSLK; and (SEQ ID NO: 1207) RWLVP.

These specific sequences are listed by way of example and they are notintended to be limiting on the scope of the present invention. In somepreferred embodiments the oligopeptidic compound has or comprises thesequence RWLVK (SEQ ID NO: 28) or KFIVK (SEQ ID NO: 119). In stillfurther embodiments, the oligopeptidic compound has or comprises thesequence RWLTK (SEQ ID NO: 76), RFLSK (SEQ ID NO: 41), RFSLK (SEQ ID NO:1206), RWLSK (SEQ ID NO: 40), RWSVK (SEQ ID NO: 52) RWAVK (SEQ ID NO:58) or RWLVP (SEQ ID NO: 1207).

Whilst the PCNA interacting motifs listed above are preferred motifs ofthe invention, in some embodiments any one or more of these motifs maybe excluded, e.g. any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or moremotifs may be excluded, such as any 25, 30, 40, 50 or more motifs or anyinteger in this range. Thus, in some embodiments the oligopeptidiccompound does not have or comprise a sequence selected from any one ormore of SEQ ID NOs: 22-297, 1206 and 1207.

Particular PCNA interacting motifs that may be excluded or disclaimedinclude any one or more of the following: KYMVR (SEQ ID NO: 298), KFLAK(SEQ ID NO: 158), KWLIK (SEQ ID NO: 268), KFLIK (SEQ ID NO: 269),KWLIOrn (SEQ ID NO: 299), KWLIDbu (SEQ ID NO: 300), and KWQLR (SEQ IDNO: 301). The oligopeptidic compound is preferably an isolated compound,e.g. an isolated peptide and most preferably the oligopeptidic compoundis a synthetic compound, e.g. a synthetic peptide. The nucleic acidmolecule encoding the oligopeptidic compound is preferably an isolatednucleic acid molecule and most preferably the nucleic acid molecule is asynthetic nucleic acid molecule. In other words, the oligopeptidiccompound and its encoding nucleic acid molecule are non-native, i.e.non-naturally occurring, molecules.

The domain that facilitates the uptake of the oligopeptidic compound maybe an uptake (import) peptide sequence, which may be a sequence whichacts to transport the oligopeptidic compound into a cell, or across acell membrane (i.e. into the interior of a cell). It may thus be aso-called “cell penetrating” sequence (or more particularly “cellpenetrating peptide”) also known in the art as a protein transductiondomain (PTD) or protein transduction sequence.

Accordingly, as noted above the invention may provide an agent orconstruct comprising (i) an oligopeptidic compound comprising an APIMmotif (i.e. a PCNA-interacting motif) as defined herein, and (ii) a cellpenetrating sequence (more particularly a cell penetrating peptide).

Cell penetrating peptide (CPP) technology has developed greatly overrecent years and a wide variety of cell penetrating peptides are knownand described in the art and indeed a range of such peptides arecommercially available. Cell penetrating peptides may vary greatly insize, sequence and charge, and indeed in their mechanism of function(which is presently not known for some peptides and not fully elucidatedfor others), but share the common ability to translocate across theplasma membrane and deliver an attached or associated moiety (theso-called “cargo”) into the cytoplasm of a cell. CPPs are thuspeptide-based delivery vectors.

Whilst CPPs are not characterized by a single structural or functionalmotif, tools to identify CPPs are available and the skilled person canreadily determine whether a peptide sequence may function to facilitatethe uptake of the peptide of which it forms a domain, i.e. whether apeptide sequence may function as an uptake (import) peptide, e.g. a CPP.For example, Hansen et al (Predicting cell-penetrating peptides,Advanced Drug Delivery Reviews, 2008, 60, pp. 572-579), provides areview of methods for CPP prediction based on the use of principalcomponent analysis (“z-predictors”) and corresponding algorithms basedon original work by Hällbrink et al (Prediction of Cell-PenetratingPeptides, International Journal of Peptide Research and Therapeutics,2005, 11(4), pp. 249-259). In brief, the methodology works by computingz-scores of a candidate peptide as based on a numerical value and anassociate range. If the z-scores fall within the range of known CPPz-scores, the examined peptides are classified as CPPs. The method wasshown to have high accuracy (about 95% prediction of known CPPs).

Additional methods for the prediction of CPPs have been developedsubsequently (see e.g. Sanders et al., Prediction of Cell PenetratingPeptides by Support Vector Machines, PLOS Computational Biology, 2011,7(7), pp. 1-12, herein incorporated by reference) and a CPP database isavailable (Gautam et al., CPPSite: a curated database of cellpenetrating peptides, Database, 2012, Article ID bas015 andhttp://crdd.osdd.net/raghava/cppsite/index.php, both herein incorporatedby reference). Accordingly, any suitable CPP may find utility in theinvention and, as discussed below, a variety of CPPs have already beenidentified and tested and could form the basis for determining andidentifying new CPPs.

CPPs may be derived from naturally-occurring proteins which are able totranslocate across cell membranes such as the Drosophila homeoboxprotein Antennapedia (a transcriptional factor), viral proteins such asthe HIV-1 transcriptional factor TAT and the capsid protein VP22 fromHSV-1, and/or they may be synthetically-derived, e.g. from chimericproteins or synthetic polypeptides such as polyarginine. As noted above,there is not a single mechanism responsible for the transduction effectand hence the design of CPPs may be based on different structures andsequences. Cell penetrating peptides are also reviewed in Jarver et al.2006 (Biochimica et Biophysica Acta 1758, pages 260-263) and Table 1below lists various representative peptides. U.S. Pat. No. 6,645,501(herein incorporated by reference) further describes various cellpenetrating peptides which might be used.

TABLE 1 CPP SEQUENCE REFERENCE Antp Class Penetratin RQIKIWFQNRRMKWKKBolton (2000) (SEQ ID NO: 302) Eur. J. Neuro. 12:287 PenatratinRRMKWKK (SEQ ID NO: 303) US 6472507 derivativesNRRMKWKK (SEQ ID NO: 304) EP4855781 QNRRMKWKK (SEQ ID NO: 305)WO 97/12912 FQNRRMKWKK (SEQ ID NO: 306) RREKWKK (SEQ ID NO: 307)RRQKWKK (SEQ ID NO: 308) KRMKWKK (SEQ ID NO: 309)RKMKWKK (SEQ ID NO: 310) RROKWKK (SEQ ID NO: 311)RRMKQKK (SEQ ID NO: 312) RRMKWFK (SEQ ID NO: 313)RORKWKK (SEQ ID NO: 314) RRMWKKK (SEQ ID NO: 315)RRMKKWK (SEQ ID NO: 316) (using standard single aminoacid notation, ornithine (O), diaminobutyric acid (B), norleucine (N))D- rqikiwfqnrrmkwkk Rouselle, C. et Penetratin (SEQ ID NO: 317)al. (2000) Mol. Pharm 57:679 Protegrin Class Pegelin RGGRLSYSRRRFSTSTGRRouselle, C. et (SynB) (SEQ ID NO: 318) al. (2000) Mol. Pharm 57:679HIV-TAT Class HIV-TAT GRKKRRQRRRPPQ Vives E.J Biol, (SEQ ID NO: 319)Chem 1997, 272:16010 Snyder (2004) PLOS 2: 186 47-57 OF YGRKKRRQRRRPotocky et al. HIV-TAT (SEQ ID NO: 320) (2003) JBC VP22DAATATRGRSAASRPTERPRAPARSAS Elliott g. Cell RPRRVD (SEQ ID NO: 321)1997, 88:223- 233 Amphipathic peptides MAP KLALKLALKALKAALKLA Morris MC., (SEQ ID NO: 322) Nat Biotechnol. 2001, 19:1173-1176Transportan GWTLNSAGYLLGKINLKALAALAKKIL Pooga M, (SEQ ID NO: 323)FASEB J 1998, 12:67- 77 Transportan- AGYLLGKINLKALAALAKKIL Soomets U, 10(SEQ ID NO: 324) Biochim Biophys Acta 2000, 1467:165-176 KALAWEAKLAKALAKALAKHLAKALAKALK Oehike J., ACEA (SEQ ID NO: 325) BiochimBiophys Acta 1998, 1414:127-139 Pep-1 KETWWETWWTEWSQPKKKRKV Wyman(SEQ ID NO: 326) Biochemistry 1997, 36:3008-3017 Pep-2KETWFETWFTEWSQPKKKRKV (SEQ ID NO: 327) MPG GALFLGFLGAAGSTMGAWSQPKSKRKVWagstaff KM (SEQ ID NO: 328) Curr Med Chem 2006, 13:1371-1387 VectocellVKRGLKLRHVRPRVTRMDV (SEQ ID NO: 329) Coupade peptidesSRRARRSPRHLGSG* (SEQ ID NO: 330) (2005)LRRERQSRLRRERQSR* (SEQ ID NO: 331) Biochem. J. GAYDLRRRERQSRLRRRERQSR407 (SEQ ID NO: 332) *indicates addition of cys for conjugation to cargo Wr-T KETWWETWWTEWWTEWSQ-GPG-rrrrrrrrKondo (2004) transporter (SEQ ID NO: 333) Mol. Can. r =D-enantiomer arginine Thera 1623 Other peptides R7RRRRRRR (SEQ ID NO: 334) Rothbard et al., Nat. Med 6 (2000) 1253- 1257

Antennapedia-derived CPPs (Antp class) represent a class of particularinterest, based around the 16 amino acid Penetratin sequence as shown inTable 1, which corresponds to the third loop of antennapedia protein andwas shown to be responsible for translocation of the protein. Penetratinhas been extensively developed as a delivery vehicle, includingparticularly for pharmaceutical use, and a wide range of Penetratinderivatives and modified sequences have been proposed and described.Reference may be made in particular to WO 91/1891, WO 00/1417, WO00/29427, WO 2004/069279 and U.S. Pat. No. 6,080,724 (hereinincorporated by reference). Thus, the 16 amino acid sequence ofPenetratin may be modified and/or truncated, or the peptide may bechemically-modified or retro-, inverso- or retro-inverso analogues maybe made whilst retaining cell-penetrating activity.

Another group of cell penetrating peptides which may advantageously beused are based on the HIV-TAT sequence and HIV-TAT and fragments thereofrepresent a preferred class of CPPs for use according to the presentinvention. Various TAT-based CPPs are described in U.S. Pat. No.5,656,122 (herein incorporated by reference). An exemplary HIV-TATpeptide as used in the Examples below is RKKRRQRRR (SEQ ID NO: 335) butit will readily be appreciated that longer or shorter TAT fragments maybe used.

As mentioned above no particular structural features or sequence motifsare common to all CPPs. However, various classes of CPPs may beidentified by particular features, such as for example peptides whichare amphipathic and net positively charged. Other groups of CPPs mayhave a structure exhibiting high α-helical content. Another group may bepeptides characterised by a high content of basic amino acids. CPPs maythus be or may comprise oligomers of basic amino acids such as argininee.g. 5 to 20, 6 to 15 or 6 to 12 R residues e.g. R₇ (SEQ ID NO: 334), R₈(SEQ ID NO: 336) or R₁₁(SEQ ID NO: 337) or QSR₈ (SEQ ID NO: 338).

Proline-rich amphipathic peptides are another class of CPP and suchpeptides characterised by the presence of pyrrolidine rings fromprolines are described in Pujals et al. 2008 Advanced Drug DeliveryReviews 60, pages 473-484 (herein incorporated by reference).

Other successfully developed CPPs include pVEC (Elmquist et al. 2003Biol. Chem 384, pages 387-393; Holm et al. 2005 Febs Lett. 579, pages5217-5222, all herein incorporated by reference) and calcitonin-derivedpeptides (Krauss et al. 2004 Bioorg. Med. Chem. Lett., 14, pages 51-54herein incorporated by reference).

Commercially available CPPs include Chariot, based on the Pep-1 peptide(Active Motif, France), the Syn-B vectors based on the protegrin peptidePG-1 (Syntem, France), and Express-si Delivery based on the MPG peptidefrom Genospectra, USA.

Other CPPs include the R41, R8, M918 and YTA-4 peptides (SEQ ID NOs:1213-1216, respectively) disclosed in Eriksson et al. 2013,Antimicrobial Agents and Chemotherapy, vol. 57(8), pp. 3704-3712(incorporated herein by reference).

In some embodiments the CPPs may be cyclic peptides, such as thosedisclosed in Oh et al., 2014, Mol. Pharmaceutics, vol. 11, pp. 3528-3536(incorporated herein by reference). In particular, the CPPs may beamphiphilic cyclic CPPs, particularly containing tryptophan and arginineresidues. In some embodiments the CPPs may be cyclic polyargininepeptides and may be modified by the addition of a fatty acyl moiety,e.g. octanoyl, dodecanoyl, hexadecanoyl,N-acetyl-L-tryptophanyl-12-aminododecanoyl etc. Suitable cyclic CPPs foruse in the invention are presented in SEQ ID NOs: 1217-1223.

In addition to publically available and reported CPPs, novel orderivative CPP peptides may be designed and synthesized based on knownor reported criteria (e.g. known CPP sequences or features such as basicamino acid content, α-helical content etc. as discussed above).Additionally, randomly-designed or other peptides may be screened forCPP activity, for example by coupling or attaching such a peptidecontaining a reporter molecule, e.g. a detectable label or tag such as afluorescent tag to the desired cargo (e.g. an oligopeptidic compound asdescribed herein) and testing to see if the construct is translocatedacross the cell membrane, for example by adding these peptides to livecells followed by examination of cellular import e.g. using confocalmicroscopy.

Indeed, whilst it is generally the case that a CPP will penetrate orenter virtually any animal cell type, it has been surprising found thatCPPs may also facilitate the uptake of peptides into microbial cells,including prokaryotic cells. It is thought that the capacity of CPPs tofunction in prokaryotic cells is a result of their structural similarityto antimicrobial peptides, e.g. short, cationic peptides withamphipathic properties. Nevertheless, it is evident from the data in theExamples that the primary anti-microbial, e.g. anti-bacterial, activityof the peptides of the invention arises from the APIM motif rather thanthe presence of a CPP sequence. It may in some cases be observed thatsuccessful or efficient delivery may be dependent, or may varydepending, on the precise nature of the cargo (e.g. cargo peptidesequence) and/or the CPP used. It would be well within the routine skillof the person skilled in the art to determine optimum peptide sequencesand combinations etc, and to test and/or modify cargo and/or CPPsequence or structure etc.

Thus, by way of summary, the skilled person will be aware of suitablecell penetrating peptide sequences that, based on the findings of theinventors, may facilitate the uptake of the oligopeptidic compound, butby way of example the sequences may include Penetratin™, a 16-amino acidpeptide corresponding to the third helix of the homeodomain ofAntennapedia protein, R rich tags such as R6-Penetratin (in whicharginine-residues were added to the N-terminus of Penetratin) andderivatives of the HIV Tat protein such as GRKKRRQRRRPPQQ (SEQ ID NO:339).

Thus, in some embodiments the domain that facilitates the cellularuptake of the oligopeptidic compound is a CPP and may be selected fromany one of:

(i) an antennapedia class peptide;

(ii) a protegrin class peptide;

(iii) a HIV-TAT class peptide;

(iv) an amphipathic class peptide selected from an amphipathic and netpositively charged peptide, a proline-rich amphipathic peptide, apeptide based on the Pep-1 peptide and a peptide based on the MPGpeptide;

(v) a peptide exhibiting high α-helical content;

(vi) a peptide comprising oligomers of basic amino acids;

(vii) pVEC;

(viii) a calcitonin-derived peptide and

(ix) an amphiphilic cyclic CPP.

In some embodiments the domain that facilitates the cellular uptake ofthe oligopeptidic compound is a CPP and may be selected from a sequenceselected from any one of SEQ ID NOs: 302-1162 or a fragment and/orderivative thereof. The details and properties of the CPPs identified inSEQ ID NOs: 340-1162 can be found athttp://crdd.osdd.net/raghava/cppsite/index.php, CPPSite: A database ofcell penetrating peptides (herein incorporated by reference).

In some embodiments the domain that facilitates the cellular uptake ofthe oligopeptidic compound is SEQ ID NO: 337.

In some embodiments, the oligopeptidic compound also comprises one ormore domains that provide a signal (target or transit) sequence. In someembodiments, the signal sequence may target the oligopeptidic compoundto a specific cell type. Additionally or alternatively, in someembodiments the oligopeptidic compound may comprise a signal peptidethat localises the compound to a specific intracellular compartment,e.g. the nucleus. In some embodiments, the uptake (import) peptide, e.g.CPP, may be sufficient to direct or localise the oligopeptidic compoundto the appropriate cellular location.

The signal sequence or signal sequence domain may thus be viewed as anysequence which acts to localise, or alternatively put, to direct,translocate or transport, the oligopeptidic compound to any desiredlocation e.g. to any desired cell type, e.g. prokaryotic or eukaryotic,or subcellular location, e.g. nucleus.

As mentioned above, in some embodiments the oligopeptidic compound (orconstructs) for use in the use and methods of the invention may compriseone or more signal sequences (i.e. one or more domains that function assignal sequences), e.g. a signal peptide which directs the compound (orconstruct) into a particular sub-cellular compartment, such as thenucleus. Nuclear localisation signals (NLSs) are again well known in theart and widely described in the literature. For instance, a searchabledatabase of known and predicted NLSs is available, see e.g. Cokol et al(Finding nuclear localization signals, EMBO Reports, 2000, 1(5), pp.411-415, herein incorporated by reference). The PSORT II database,http://psort.hgc.jp/ (herein incorporated by reference) can be used forthe prediction of nuclear localization of proteins based on NLSs.Accordingly, any known or functional NLS may find utility in theinvention.

An NLS may vary in length and/or sequence and a wide range of specificNLS sequences have been described. In general, however, it has beenfound that peptides comprising positively charged amino acids (notablylysine (K), arginine (R) and/or histidine (H)) may function as an NLS.An exemplary NLS may thus be a peptide of e.g. 4-20, more particularly4-15, 4-12, 4-10 or 4-8 amino acids, wherein at least 4 amino acids (andmore particularly at least 60, 70, 75, 80, 85, or 90% of the amino acidresidues in the NLS peptide) are positively charged amino acids,preferably selected from K, R or H. Such an exemplary NLS may forexample have or comprise the sequence RKRH (SEQ ID NO: 1163).

Nuclear localisation signals, including both actualexperimentally-determined and predicted or proposed NLS sequences, andstrategies for identifying NLSs are also described in Lange et al., J.Biol. Chem. 2007, 282(8), 5101-5105; Makkerh et al., Current Biology1996, 6(8), 1025-1027; Leslie et al., Methods 2006, 39, 291-308; andLusk et al. Nature Reviews MCB 2007, 8, 414-420 (all herein incorporatedby reference).

A classical NLS consists of either one (monopartite) or two (bipartite)stretches of basic amino acids. A monopartite NLS may be exemplified bythe SV40 large T antigen NLS (¹²⁶PKKKRKV¹³² [SEQ ID NO: 1164]) and abipartite NLS by the nucleoplasmin NLS (¹⁶⁶ KRPAATKKAGQAKKKK¹⁷⁶[SEQ IDNO: 1165]). The monopartite NLS consensus sequence K-[K/R]-X-[K/R] (SEQID NO: 1166) has been proposed and accordingly an NLS according to thepresent invention may in one embodiment comprise or consist of such aconsensus sequence (where X is any amino acid).

A representative bipartite NLS according to the invention may have thesequence KR-[X]₅₋₂₀-KKKK (SEQ ID NO: 1167), e.g. KR-X₁₀-KKKK (SEQ ID NO:1168) (where X is any amino acid).

An alternative exemplary bipartite NLS may take the form RKRH-[X]₂₋₁₀-KK(SEQ ID NO: 1169) e.g. RKRH-X₂-KK (SEQ ID NO: 1170), for exampleRKRH-II-KK (SEQ ID NO: 1171).

The oncoprotein c-myc NLS differs from classical NLSs in that only 3 of9 amino acid residues are basic (PAAKRVKLD [SEQ ID NO: 1172]),indicating that an NLS need not necessarily conform to the consensus orclassical sequences given above. Makkerh et al (supra) describe NLSsequences in which a cluster of basic amino acids (e.g. KKKK [SEQ ID NO:1173]) is flanked by neutral and acidic residues, for example PAAKKKKLD(SEQ ID NO: 1174).

Other possible NLS sequences which may be given by way of exampleinclude: PKKKRKVL (SEQ ID NO: 1175), KKKRK (SEQ ID NO: 1176), KKKRVK(SEQ ID NO: 1177), KKKRKVL (SEQ ID NO: 1178) and RKKRKVL (SEQ ID NO:1179). Any NLS which is a derivative of a known NLS e.g. the SV40,nucleoplasmin, UNG2 or c-myc NLS may be used.

A putative, proposed or predicted NLS sequence can be tested for NLSactivity using principles and assays known and described in the art. Forexample a candidate NLS sequence may be attached to the desired cargo(in this case an oligopeptidic compound as defined herein) and theconstruct may be provided with a detectable reporter molecule (e.g. atag or label which may be visualised, for example a fluorescent label)and contacted with a test cell. Distribution of the construct in thecell may then be determined.

Thus, by way of summary, the skilled person will be aware of suitablesignal sequences, but by way of example the following are mentionedherein Examples of nuclear localisation sequences include the SV40protein derivative KKKRK (SEQ ID NO: 1176).

Thus, in some embodiments the oligopeptidic compound comprises a signalsequence (i.e. a domain comprising a signal peptide) that localizes ordirects the oligopeptidic compound to a sub-cellular location, such as aNLS and may be selected from any one of:

(i) a peptide of 4-20 amino acids, wherein at least 4 amino acids arepositively charged amino acids, preferably selected from K, R or H;and/or

(ii) a sequence selected from any one of SEQ ID NOs: 1163-1179 or afragment and/or derivative thereof.

In some embodiments the nuclear localisation signal sequence comprises asequence selected from any one of SEQ ID NOs: 1163-1179 or a fragmentand/or derivative thereof, preferably wherein said fragment and/orderivative comprises at least 4 positively charged amino acids,preferably selected from any of K, R or H.

In some embodiments an oligopeptidic compound or construct according tothe present invention may comprise at least three domains, including (i)an APIM motif domain as defined herein, (ii) a linker domain, which mayin some embodiments comprise a nuclear localisation signal sequence, and(iii) a peptide sequence domain that facilitates the cellular uptake ofsaid compound or construct (i.e. an uptake/import peptide sequencedomain, e.g. cell penetrating signal sequence domain).

The separate elements or components (domains) of a construct accordingto the present invention may be contained or presented in any order, butpreferably in the orders indicated above (e.g. APIM oligopeptidiccompound-CPP or APIM oligopeptidic compound-linker-CPP).

In some embodiments, the APIM motif is located at or towards theN-terminus of the peptide. For instance, the APIM motif may be describedas being N-terminal to the peptide sequence domain that facilitates thecellular uptake of said compound (e.g. the CPP) and optionallyN-terminal to the linker sequence, if present.

In a preferred embodiment, the oligopeptidic compound comprises a PCNAinteracting motif as set forth in SEQ ID NO: 28, a nuclear localisationsignal sequence/linker sequence as set forth in SEQ ID NO: 1176 and acell penetrating signal sequence as set forth in SEQ ID NO: 337.

Furthermore, in some embodiments an oligopeptidic compound or constructaccording to the invention may contain more than one PCNA-interactingmotif. Thus, alternatively put, an agent for use in the uses and methodsof the present invention may contain or encode an oligopeptidic compoundcomprising more than one PCNA-interacting motif. A construct oroligopeptidic compound may for example contain 1-10, e.g. 1-6, or 1-4 or1-3 or one or two motifs. Within a construct also containing a signalsequence, such motifs may be spaced or located according to choice, e.g.they may be grouped together, or they may be separated by signalsequence elements e.g. motif-motif-CPP, motif-linker-motif-CPP; ormotif-linker-motif-motif-CPP; or motif-motif-linker-CPP etc.

As referred to herein a “fragment” may comprise at least 30, 40, 50, 60,70, 80, 85, 90, 95, 96, 97, 98 or 99% of the amino acids of the sequencefrom which it is derived. Said fragment may be obtained from a centralor N-terminal or C-terminal portions of the sequence. Whilst the size ofthe fragment will depend on the size of the original sequence, in someembodiments the fragments may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15 or more amino acid residues shorter than the sequence fromwhich it is derived, e.g. 1-10, 2-9, 3-8, 4-7 amino acid residuesshorter than the sequence from which it is derived.

As referred to herein a “derivative” of a sequence is at least 55, 60,65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the sequenceto which it is compared.

Sequence identity may be determined by, e.g. using the SWISS-PROTprotein sequence databank using FASTA pep-cmp with a variable pamfactor,and gap creation penalty set at 12.0 and gap extension penalty set at4.0, and a window of 2 amino acids. Preferably said comparison is madeover the full length of the sequence, but may be made over a smallerwindow of comparison, e.g. less than 200, 100, 50, 20 or 10 contiguousamino acids.

Preferably such sequence identity related polypeptides, i.e.derivatives, are functionally equivalent to the peptides which are setforth in the recited SEQ ID NOs. Similarly, the peptides with sequencesas set forth in the SEQ ID NOs. may be modified without affecting thesequence of the polypeptide as described below.

Furthermore, “fragments” as described herein may be functionalequivalents. Preferably these fragments satisfy the identity (relativeto a comparable region) conditions mentioned herein.

As referred to herein, to achieve “functional equivalence” the peptidemay show some reduced efficacy in performing the function relative tothe parent molecule (i.e. the molecule from which it was derived, e.g.by amino acid substitution), but preferably is as efficient or is moreefficient. Thus, functional equivalence may relate to a peptide which iseffective in localizing or directing the oligopeptidic compound to thecell type or cellular location, e.g. to facilitate to the uptake of thepeptide as described above. This may be tested by comparison of theeffects of the derivative peptide relative to the peptide from which itis derived in a qualitative or quantitative manner, e.g. by performingthe in vitro analyses described above. Where quantitative results arepossible, the derivative is at least 30, 50, 70 or 90% as effective asthe parent peptide.

Functionally-equivalent peptides which are related to or derived fromthe parent peptide, may be obtained by modifying the parent amino acidsequence by single or multiple amino acid substitution, addition and/ordeletion (providing they satisfy the above-mentioned sequence identityrequirements), but without destroying the molecule's function.Preferably the parent sequence has less than 20 substitutions, additionsor deletions, e.g. less than 10, 5, 4, 3, 2, or 1 such modifications.Such peptides may be encoded by “functionally-equivalent nucleic acidmolecules” which may be generated by appropriate substitution, additionand/or deletion of one or more bases.

The domains (which may be viewed as components, elements or separateparts) of an oligopeptidic compound or construct as described herein maybe attached or linked to one another in any desired or convenient wayaccording to techniques well known in the art. Thus, the domains may belinked or conjugated chemically, e.g. using known chemical couplingtechnologies or the compound or constructs may be formed as a singlewhole using genetic engineering techniques e.g. techniques for formingfusion proteins, or they may simply be synthesized as a whole, e.g.using peptide synthesis techniques.

The domains may be linked directly to each other or they may be linkedindirectly by means of one or more linker (or spacer) sequences. Thus, alinker sequence may interspace or separate two or more individualdomains (i.e. parts, e.g. or separate motif elements) in anoligopeptidic construct or compound. The precise nature of the linkersequence is not critical and it may be of variable length and/orsequence, for example it may have 0-40, more particularly 0-20, 0-15,0-12, 0-10, 0-8, or 0-6, 0-4 or 0-3 residues e.g. 1, 2 or 3 or moreresidues. By way of representative example the linker sequence, ifpresent, may have 1-15, 1-12, 1-10, 1-8, 1-6 or 1-4 residues etc. Thenature of the residues is not critical and they may for example be anyamino acid, e.g. a neutral amino acid, or an aliphatic amino acid, oralternatively they may be hydrophobic, or polar or charged orstructure-forming e.g. proline. A range of different linker sequenceshave been shown to be of use, including short (e.g. 1-6) sequences ofneutral and/or aliphatic amino acids.

Exemplary linker sequences thus include any single amino acid residue,e.g. A, I, L, V, G, R, Q, T, or W, or a di-, tri- tetra- penta- orhexa-peptide composed of such residues.

As representative linkers may be mentioned I, II, IL, R, W, WW, WWW,RIL, RIW, GAQ, GAW, VAT, IILVI (SEQ ID NO: 1180), IILVIII (SEQ ID NO:1181) etc.

The linkers between different domains (components, elements or parts)may be the same or different.

As mentioned above, in some embodiments the linker may comprise orconsist of an NLS. Alternatively viewed, in some embodiments an NLS,when present, may function both as a signal peptide and a linker. Thus,the oligopeptidic compound may comprise a signal peptide (e.g. an NLS)and a linker.

Representative compounds (or more particularly constructs) for use inthe methods and uses of the invention include:

(SEQ ID NO: 1182) MDRWLVKRILVATK, (SEQ ID NO: 1183)MDRWLVKRILKKKRKVATKG, (SEQ ID NO: 1184)MDRWLVKGAQPKKKRKVLRQIKIWFQNRRMKWKK, (SEQ ID NO: 1185)MDRWLVKGAWKKKRVKIIRKKRRQRRRK, (SEQ ID NO: 1186)MDRWLVKGAWKKKRKIIRKKRRQRRRG, (SEQ ID NO: 1187)MDRWLVKGAWKKKRKIIRKKRRQRRRK, (SEQ ID NO: 1188)MDRWLVKRIWKKKRKIIRKKRRQRRRK, (SEQ ID NO: 1189)MDRWLVKWWWKKKRKIIRKKRRQRRRK, (SEQ ID NO: 1190)MDRWLVKWWRKRHIIKKRKKRRQRRRK, (SEQ ID NO: 1191)MDRWLVKRIWKKKRKIIRRRRRRRRRRRK, (SEQ ID NO: 1192)MDRWLVKRIWKKKRKIIRQIKIWFQNRRMKWKK, (SEQ ID NO: 1193)MDRFLVKGAWRKRHIIKKRKKRRQRRRK, (SEQ ID NO: 1194)MDRWLVKWKKKRKIRRRRRRRRRRRK, (SEQ ID NO: 1195) MDRWLVKWKKKRKIRKKRRQRRRK,(SEQ ID NO: 1196) MDRWLVKWRKRHIRKKRRQRRRK, (SEQ ID NO: 1197)Ac-MDRWLVKGAWRKRHIRKKRRQRRRK, (SEQ ID NO: 1198)Ac-MDRWLVKWKKKRKIRRRRRRRRRRR, (SEQ ID NO: 1199)Ac-MDRFLVKWKKKRKIRRRRRRRRRRR, (SEQ ID NO: 1200)Ac-MDRWLVKKKKRKRRRRRRRRRRRK, (SEQ ID NO: 1201)Ac-MDRWLVKKKKRKRRRRRRRRRRR, (SEQ ID NO: 1202)MDRWLVKRIWKKKRKIIRWLVKWWWRKKRRQRRRK (SEQ ID NO: 1203)MDRWSVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1204) MDRWAVKWKKKRKIRRRRRRRRRRR(SEQ ID NO: 1208) MDRWLTKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1209)MDRFSLKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1210) MDRFLSKWKKKRKIRRRRRRRRRRR(SEQ ID NO: 1211) MDRWLSKWKKKRKIRRRRRRRRRRR or (SEQ ID NO: 1212)MDRWLVPWKKKRKIRRRRRRRRRRR .

In a particularly preferred embodiment, the oligopeptidic compoundcomprises a sequence as set forth in SEQ ID NO: 1198, 1203, 1204 or 1208to 1212. The oligopeptidic compounds shown above comprise N-terminalamino acids that do not form part of the domains that are essential forthe compounds to have activity in the methods and uses of the invention,i.e. an “MD” sequence. Some of the peptides also comprise N-terminalmodification, e.g. acetyl groups. These additional amino acids andmodifications may facilitate the production of the oligopeptidiccompounds, e.g. in vitro or in vivo, and/or help to protect thecompounds from degradation in vivo. It will be evident that theoligopeptidic compounds do not require these additional amino acids ormodifications for their activity. Accordingly, further representativesequences according to the invention include any of SEQ ID NOs: 1182 to1204 or 1208 to 1212, omitting the N-terminal “MD” and/or “Ac” groups.In other embodiments, a C-terminal K or G residue may additionally oralternatively be omitted. Furthermore, the presence of additional aminoacids or modifications at either terminus would not be expected todisrupt or inhibit the function of the oligopeptidic compounds describedherein. Thus, in some embodiments, the oligopeptidic compound maycomprise an N-terminal sequence, e.g. a sequence at the N-terminus thatdoes not comprise a domain defined above, e.g. a so-called N-terminalflanking sequence. In some embodiments, the oligopeptidic compound maycomprise a C-terminal sequence, e.g. a sequence at the C-terminus thatdoes not comprise a domain defined above, e.g. a so-called C-terminalflanking sequence. In some embodiments, the oligopeptidic compound maycomprise an N-terminal and C-terminal flanking sequence.

A flanking sequence may comprise from about 1-150 amino acids, such as1-120, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-35, 1-30 etc. Thus,a flanking sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids, e.g.1-40, 2-39, 3-38, 4-37, 5-36, 6-35, 7-34, 8-33, 9-32, 10-31, 11-30,12-29, 13-28, 14-27, 15-26 amino acids or any combination thereof.

Oligopeptidic compounds having sequences as set out in SEQ ID NOs.1182-1204 and 1208-1212 comprise separate domains (i.e. components)making up the constructs (i.e. motif-containing sequence, linker/NLS,CPP, etc.) Thus, it will be seen that SEQ ID NOs. 1182-1204 and1208-1212 represent constructs comprising at least one motif-containingsequence, a linker/NLS and a CPP, in some cases linked by linkersequences which may vary in sequence, as specified. NLS sequences basedon the SV40 or UNG2 NLS sequences are used, and CPP sequences based onPenetratin, HIV-TAT or an R-rich peptide.

The standard amino acid one letter code is used herein, so K stands forlysine (Lys), I stands for isoleucine (Ile) and so on.

As mentioned above, the oligopeptidic compound, and more particularly,the APIM motif, may comprise non-conventional or non-standard aminoacids. Other domains in the oligopeptidic compound may also incorporatenon-standard amino acids. In some embodiments, the oligopeptidiccompound may comprise one or more, e.g. at least 1, 2, 3, 4 or 5non-conventional amino acids, i.e. amino acids which possess a sidechain that is not coded for by the standard genetic code, termed herein“non-coded amino acids” (see e.g. Table 2). These may be selected fromamino acids which are formed through metabolic processes such asornithine or taurine, and/or artificially modified amino acids such as9H-fluoren-9-ylmethoxycarbonyl (Fmoc), (tert)-(B)utyl (o)xy (c)arbonyl(Boc), 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc) protected aminoacids, or amino acids having the benzyloxy-carbonyl (Z) group.Preferably, where such non-coded amino acids are present, they are notlocated within the motif, but in some embodiments one or more non-codedamino acids are present within the motif. In some embodiments, non-codedamino acids are present in more than one domain of the oligopeptidiccompound.

In vitro and/or in vivo stability of the oligopeptidic compound may beimproved or enhanced through the use of stabilising or protecting meansknown in the art, for example the addition of protecting or stabilisinggroups, incorporation of amino acid derivatives or analogues or chemicalmodification of amino acids, Such protecting or stabilising groups mayfor example be added at the N and/or C-terminus. An example of such agroup is an acetyl group and other protecting groups or groups whichmight stabilise a peptide are known in the art.

The oligopeptidic compounds of the invention will typically compriseonly amino acids having the L-configuration, but one or more amino acidshaving the D configuration may be present. In some embodiments theoligopeptidic compound contains at least 1, 2, 3, 4 or 5 D-amino acidsand they are preferably found in the motif, but in another embodiment,D-amino acids are present only outside of the motif. In a still furtherembodiment, D-amino acids may be found in more than one domain of theoligopeptidic compound. The oligopeptidic compound may be linear orcyclic.

Thus, included particularly are retro-inverso oligopeptidic compounds ofthe oligopeptidic compounds of the invention (and more particularlyretro-inverso peptides). Retro-inverso oligopeptidic compounds compriseD-amino acids in reverse (opposite) order to the parental or referencecompound sequence. A retro-inverso analogue thus has reversed terminiand reversed order of e.g. peptide bonds, while approximatelymaintaining the topology of the side chains as in the parental orreference sequence.

The oligopeptidic compound may include partial retro-inverso sequences,i.e. a domain or part of a domain may comprise a retro-inverso sequence.

By “oligopeptidic compound” is meant a compound which is composed ofamino acids or equivalent subunits, which are linked together by peptideor equivalent bonds. Thus, the term “oligopeptidic compound” includespeptides and peptidomimetics.

By “equivalent subunit” is meant a subunit which is structurally andfunctionally similar to an amino acid. The backbone moiety of thesubunit may differ from a standard amino acid, e.g. it may incorporateone or more nitrogen atoms instead of one or more carbon atoms. Inpreferred embodiments, the subunit comprises a standard amino acidbackbone, i.e. the backbone of a standard or coded amino acid. In otherwords, preferably the subunit is an amino acid. However, the amino acidsubunit may comprise a non-standard (non-coded) R-group.

By “peptidomimetic” is meant a compound which is functionally equivalentor similar to a peptide and which can adopt a three-dimensionalstructure similar to its peptide counterparts, but which is not solelycomposed of amino acids linked by peptide bonds. A preferred class ofpeptidomimetics are peptoids, i.e. N-substituted glycines. Peptoids areclosely related to their natural peptide counterparts, but they differchemically in that their side chains are appended to nitrogen atomsalong the molecule's backbone, rather than to the α-carbons as they arein amino acids.

Peptidomimetics, particularly non-peptidic molecules may be generatedthrough various processes, including conformational-based drug design,screening, focused library design and classical medicinal chemistry. Notonly may oligomers of unnatural amino acids or other organic buildingblocks be used, but also carbohydrates, heterocyclic or macrocycliccompounds or any organic molecule that comprises structural elements andconformation that provides a molecular electrostatic surface that mimicsthe same properties of the 3-dimensional conformation of the peptide maybe used by methods known in the art.

Thus the peptidomimetics may bear little or no resemblance to a peptidebackbone. Peptidomimetics may comprise an entirely synthetic non-peptideform (e.g. based on a carbohydrate backbone with appropriatesubstituents) or may retain one or more elements of the peptide on whichit is based, e.g. by derivatizing one or more amino acids or replacingone or more amino acids with alternative non-peptide components.Peptide-like templates include pseudopeptides and cyclic peptides.Structural elements considered redundant for the function of the peptidemay be minimized to retain a scaffold function only or removed whereappropriate.

In preferred embodiments, peptidomimetics retain one or more peptideelements, i.e. more than one amino acid, although such amino acids maybe replaced with a non-standard or structural analogue thereof. Aminoacids retained in the sequences may also be derivatised or modified(e.g. labelled, glycosylated or methylated) as long as the functionalproperties of the oligopeptidic compound are retained. Thepeptidomimetics are referred to as being “derivable from” a certainpolypeptide sequence. By this it is meant that the peptidomimetic isdesigned with reference to the peptide sequence defined above, such thatit retains the structural features of the peptide which are essentialfor its function. This may be the particular side chains of the peptide,or hydrogen bonding potential of the structure. Such features may beprovided by non-peptide components or one or more of the amino acidresidues or the bonds linking said amino acid residues of thepolypeptide may be modified so as to improve certain functions of thepeptide such as stability or protease resistance, while retaining thestructural features of the peptide which are essential for its function.

Examples of non-standard or structural analogue amino acids which may beused are D amino acids, amide isosteres (such as N-methyl amide,retro-inverse amide, thioamide, thioester, phosphonate, ketomethylene,hydroxymethylene, fluorovinyl, (E)-vinyl, methyleneamino, methylenethioor alkane), L-N methylamino acids, D-α methylamino acids,D-N-methylamino acids. Examples of non-conventional, i.e. non-coded,amino acids are listed in Table 2.

TABLE 2 Non-conventional Non-conventional amino acid Code amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycinecarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbc L-O-methyl serineOmser ethylamino)cyclopropane L-O-methyl homoserine Omhser

In preferred embodiments, the oligopeptidic compound is a peptide. Inparticularly preferred embodiments, the oligopeptidic compound is apeptide consisting of L-amino acids. In yet a further preferredembodiment, the oligopeptidic compound is a peptide consisting ofstandard or coded L-amino acids.

As mentioned above, the oligopeptidic compound may comprise non-standardamino acids. Thus, in some embodiments the oligopeptidic compound mayincorporate di-amino acids and/or β-amino acids. However, in preferredembodiments, at least the APIM motif domain, consists of α-amino acids.Most preferably, the oligopeptidic compound, i.e. all domains andoptionally all flanking sequences, consists of α-amino acids.

As mentioned above, the oligopeptidic compound defined herein comprisesmore than 5 subunits, but the length of the construct will depend on thesize of the uptake peptide sequence and on the number and size of otherdomains, e.g. linker domains, flanking sequences etc, if present. Thus,the prefix “oligo” is used to designate a relatively small number ofsubunits such as amino acids, i.e. less than 200, preferably less than150, 100, 90, 80, 70, 60 or 50 subunits. The oligopeptidic compound ofthe invention may thus comprise more than 5 but no more than 200subunits. Preferably, it comprises at least 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25 subunits. Alternatively defined itcomprises no more than 50, 45, 40, 35, 34, 33, 32, 31 or 30 subunits.Representative subunit ranges thus include 12-50, 12-45, 12-40, 12-35,12-30, 12-25, 12-22, 12-20, 12-18 etc, 12-30 and 12-40 being preferred.

The nature of the subunits of the oligopeptidic compound outside of theAPIM motif domain and the uptake peptide sequence is not critical, sothe subunits outside of the motif may for example be those found in anative protein comprising the motif, such as hABH2, or they may bealanine residues or any other suitable residues.

Peptidomimetics typically have a longer half life within a patient'sbody, so they may be preferred in embodiments where a longer lastingeffect is desired. This can help reduce the frequency at which thecomposition has to be re-administered. Furthermore, peptidomimetics maybe particularly useful in the in vitro methods described herein.However, for bio-safety reasons a shorter half life may be preferred inother embodiments; in those embodiments peptides are preferred.

The oligopeptidic compound may form part of a larger unit, e.g. it maybe fused to a polypeptide to form a recombinant fusion protein orattached to a scaffold to form a peptide aptamer. Thus, fusion proteinsor aptamers incorporating the oligopeptidic compound may also findutility in the uses and methods of the invention, i.e. in someembodiments the agent may be a fusion protein or aptamer comprising theoligopeptidic compound defined above.

Yet further aspects include pharmaceutical compositions comprising theagent defined herein, e.g. comprising the oligopeptidic compound, fusionprotein or aptamer, together with at least one pharmacologicallyacceptable carrier or excipient, wherein said composition is for use inthe uses and methods of the invention defined below.

In a further aspect, a nucleic acid molecule encoding a peptide havingor comprising (e.g. of) SEQ ID NO: 1, as defined above, is provided foruse in the methods and uses of the invention. Alternatively viewed, theagent or composition for use in the uses and methods of the inventionmay be a nucleic acid molecule encoding a peptide having or comprising(e.g. of) SEQ ID NO: 1, as defined above. In this respect, the nucleicacid molecule may not need to encode all of the domains of theoligopeptidic compound described above, e.g. the domain that facilitatesthe cellular uptake of the peptide. For instance, the nucleic acidmolecule may be delivered into the cell by another mechanism, e.g. via aliposome. However, in a preferred embodiment, the invention provides anucleic acid molecule encoding an oligopeptidic compound or construct(e.g. a peptide) as defined above, comprising a PCNA interacting motif(APIM motif) domain and a peptide sequence (domain) that facilitates theuptake of said peptide. Also provided is the complement of such anucleic acid molecule for use in the uses and methods of the invention.Thus, in some embodiments the nucleic acid molecule may also encode oneor more linker and/or signal sequences, as defined above.

The nucleic acid molecule of the invention comprises at least 15nucleotides, preferably at least 36 nucleotides, and preferably no morethan 800 nucleotides, more preferably no more than 700, 650, 600, 550,500, 450, 400, 350, 300, 250, 200, 150, 100, 75 or 50 nucleotides. Thenucleic acid molecule is preferably an isolated or synthetic molecule.

A further aspect relates to a vector comprising a nucleic acid moleculeas defined herein for use in the uses and methods defined below.Preferably, the vector comprises a promoter sequence operably linked tothe sequence encoding a peptide as defined above. The vector may alsocontain further elements typically found in a vector such as an originof replication, a selectable marker such as antibiotic resistance,and/or a multiple cloning site. The vector may further be an expressionvector, and may comprise further elements, e.g. transcriptional and/ortranslational control or regulatory elements for expression of thenucleic acid molecules. Such control elements, e.g. promoters, ribosomebinding sites, enhancers, terminators etc. are well known and widelydescribed in the art.

The vector may for example be a plasmid or a viral genome (or partthereof), preferably the viral genome is from a virus selected from aretrovirus, an adenovirus and an adenovirus-associated virus. In someembodiments, e.g. where the vector comprises a nucleic acid moleculeencoding a bactericidal peptide, the vector may be a viral genome (orpart thereof) from a virus capable of infecting a bacterium such as abacteriophage. Thus, in some embodiments, the vector may be administeredin the form of a virus comprising a vector containing a nucleic acidmolecule encoding an oligopeptidic compound described above.Alternatively viewed, in some embodiments the vector may be a virus.

As mentioned above, there is provided a composition (e.g. apharmaceutical composition) comprising an agent as defined herein foruse in the methods and uses of the invention. Accordingly, saidcomposition (e.g. a pharmaceutical composition) may comprise anoligopeptidic compound (including a fusion protein or aptamer) and/ornucleic acid molecule as defined herein and/or a vector as definedherein, together with at least one pharmacologically (orpharmaceutically) acceptable carrier or excipient.

The excipient may include any excipients known in the art, for exampleany carrier or diluent or any other ingredient or agent such as buffer,antioxidant, chelator, binder, coating, disintegrant, filler, flavour,colour, glidant, lubricant, preservative, sorbent and/or sweetener etc.

The excipient may be selected from, for example, lactic acid, dextrose,sodium metabisulfate, benzyl alcohol, polyethylene glycol, propyleneglycol, microcrystalline cellulose, lactose, starch, chitosan,pregelatinized starch, calcium carbonate, calcium sulfate, dextrates,dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calciumphosphate, magnesium carbonate, magnesium oxide, maltodextrin, mannitol,powdered cellulose, sodium chloride, sorbitol and/or talc.

The pharmaceutical composition may be provided in any form known in theart, for example as a tablet, capsule, coated tablet, liquid,suspension, tab, sachet, implant, inhalant, powder, pellet, emulsion,lyophilisate, effervescent, spray, salve, emulsion, balm, plaster or anymixtures thereof. It may be provided e.g. as a gastric fluid-resistantpreparation and/or in sustained action form. It may be a form suitablefor oral, parenteral, topical, rectal, genital, subcutaneous,transurethral, transdermal, intranasal, intraperitoneal, intramuscularand/or intravenous administration and/or for administration byinhalation. In embodiments where the composition is used in combinationwith UV radiotherapy, the composition preferably may be formulated fortopical administration, e.g. for the treatment or prevention of abacterial infection in a wound, including a surgical wound.

In a representative embodiment, the pharmaceutical composition may be ina form suitable for liposomal administration, so preferably liposomescontaining the pharmaceutical composition are provided. When liposomesare used, it may not be necessary to include a further excipient, soalso provided are liposomes containing an agent, e.g. oligopeptidiccompound, as defined herein, for use in the methods and uses of theinvention.

The term “treatment” as used herein refers broadly to any effect or step(or intervention) beneficial in the management of a clinical conditionor disorder and thus includes both therapeutic and prophylactictreatments. Treatment may include reducing, alleviating, ameliorating,slowing the development of, or eliminating the condition or one or moresymptoms thereof, which is being treated, relative to the condition orsymptom prior to the treatment, or in any way improving the clinicalstatus of the subject. A treatment may include any clinical step orintervention which contributes to, or is a part of, a treatmentprogramme or regimen. A prophylactic treatment may include delaying,limiting, reducing or preventing the condition or the onset of thecondition, or one or more symptoms thereof, for example relative to thecondition or symptom prior to the prophylactic treatment. Prophylaxisthus explicitly includes both absolute prevention of occurrence ordevelopment of the condition, or symptom thereof, and any delay in theonset or development of the condition or symptom, or reduction orlimitation on the development or progression of the condition orsymptom.

Thus, treatment includes killing, inhibiting or slowing the growth ofmicrobial cells, or the increase in size of a body or population ofmicrobial cells, reducing microbial cell number or preventing the spreadof microbial cells (e.g. to another anatomic site), reducing the size ofa microbial cell colony or infection site etc. The term “treatment” doesnot necessarily imply the cure or complete abolition or elimination ofmicrobial cell growth, or growth of microbial cells.

The term “inhibit” is used broadly to include any reduction or decreasein microbial cell growth as well as the prevention or abolition ofmicrobial cell growth. “Inhibition” thus includes the reduction orprevention of microbial cell growth, e.g. including reducing the rate ofcell growth. This may be determined by any appropriate or convenientmeans, such as determining or assessing cell number, cell viabilityand/or cell death etc., as may be determined by techniques well known inthe art.

“Growth” of microbial cells as referred to herein is also used broadlyto include any aspect of microbial cell growth, including in particularthe proliferation (i.e. increase in number) of microbial cells.

The agents as defined herein may thus be used in the treatment orprevention of any microbial infection, which may be a disease orcondition (used broadly herein to include any disorder or any clinicalsituation) which is responsive to reduction of microbial cell growth(particularly microbial cell proliferation). The agents accordingly findutility in any therapy (or treatment) which targets microbial cellgrowth (or proliferation). In other words, the agents may be used in anytherapeutic application in which it desirable or advantageous to inhibitmicrobial cell proliferation.

A “microbial infection” may be defined as any atypical, unwanted,undesirable, excessive and/or harmful infection and includes a“microbial infectious disease” and may be defined as a disease,condition or disorder caused by the invasion of a subject, e.g. one ormore organs or tissues of said subject, by one or more disease-causingmicroorganisms and their subsequent multiplication. In some instances,an infection or infectious disease may be characterised by the reactionof the subject (e.g. organ or tissues of said subject) to said organismsand, in some cases, to the toxins produced by said organisms. Amicrobial infection or microbial infectious disease may be local orsystemic. A microbial infection may be any bacterial or fungalinfection, i.e. caused by a bacterium or fungus.

Since the therapeutic applications and utilities of the presentinvention may generally involve inhibiting microbial cell proliferation,any proliferating microbial cell may be targeted in the therapies andutilities disclosed and encompassed herein. Such proliferating microbialcells may include bacterial and/or fungal cells.

In some aspects of the invention the bacteria may be a gram positive orgram negative, or gram test non-responsive. They may be aerobic oranaerobic bacteria. For instance, the bacteria may be from any of thegenus Acinetobacter, Bacillus, Burkholderia, Chlamydia, Clostridium,Helicobacter, Staphylococcus, Streptococcus, Pseudomonas, Legionella,Listeria, Mycobacterium, Proteus, Klebsiella, Fusobacterium or otherenteric or coliform bacteria.

Thus, for instance, the microbial infection or microbial infectiousdisease (e.g. bacterial infection or bacterial infectious disease) maybe caused by a gram-positive bacterium such as, M. tuberculosis, M.bovis, M. typhimurium, M. bovis strain BCG, BCG substrains, M. avium, M.intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M.avium subspecies paratuberculosis, Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus equi, Streptococcus pyogenes, Streptococcusagalactiae, Listeria monocytogenes, Listeria ivanovii, Bacillusanthracis, B. subtilis, Nocardia asteroides, Actinomyces israelii,Propionibacterium acnes, and Enterococcus species.

In other embodiments, the microbial infection or microbial infectiousdisease (e.g. bacterial infection or bacterial infectious disease) maybe caused by a gram-negative bacterium such as Clostridium tetani,Clostridium perfringens, Clostridium botulinum, Pseudomonas aeruginosa,Vibrio cholerae, Actinobacillus pleuropneumoniae, Pasteurellahaemolytica, Pasteurella multocida, Legionella pneumophila, Salmonellatyphi, Brucella abortus, Chlamydi trachomatis, Chlamydia psittaci,Coxiella burnetti, Escherichia coli, Neiserria meningitidis, Neiserriagonorrhea, Haemophilus influenzae, Haemophilus ducreyi, Yersinia pestis,Yersinia enterolitica, Escherichia coli, E. hirae, Burkholderia cepacia,Burkholderia pseudomallei, Francisella tularensis, Bacteroides fragilis,Fusobascterium nucleatum, and Cowdria ruminantium.

In some embodiments, the microbial infection or microbial infectiousdisease may be caused by a bacterium is selected from the followinggenera: Achromobacter, Acinetobacter, Actinobacillus, Aeromonas,Agrobacterium, Alcaligenes, Alteromonas, Bacteroides, Bartonella,Borrelia, Bordetella, Brucella, Burkholderia, Campylobacter,Cardiobacterium, Chlamydia, Chlamydophila, Chromobacterium,Chyseobacterium, Chryseomonas, Citrobacter, Clostridium, Comamonas,Corynebacterium, Coxiella, Cryptobacterium, Edwardsiella, Eikenella,Enterobacter, Enterococcus, Erwinia, Helicobacter, Kingella, Klebsiella,Lactobacillus, Lactococcus, Legionella, Leptospira, Leptotrichia,Leuconostoc, Listeria, Listonella, Mobiluncus, Moraxella, Morganella,Mycobacterium, Mycoplasma, Neisseria, Nocardia, Nocardiopsis, Pantoea,Parachlamydia, Pasteurella, Peptococcus, Peptostreptococcus, Prevotella,Propionibacterium, Proteus, Providencia, Pseudomonas, Ralstonia,Rickettsia, Salmonella, Shewenella, Shigella, Sphingobacterium,Sphingomonas, Staphylococcus (e.g. Staphylococcus aureus NCTC 6571 alsocalled Oxford Staph.), Stenotrophomonas, Streptobacillus, Streptococcus,Streptomyces, Treponem and Yersinia. In some embodiments, the bacteriumis a MDR bacterium.

In some embodiments, the MDR bacterium is a Methicillin-resistantStaphylococcus aureus (MRSA) bacterium. Alternatively viewed, the MDRbacterial infection is an MRSA infection.

MRSA infections are caused by strains of Staphylococcus aureus that havebecome resistant to the antibiotics commonly used to treat ordinaryStaphylococcus aureus infections.

Most MRSA infections occur in people who have been in hospitals or otherhealth care settings, such as nursing homes and dialysis centres, whereit is known as health care-associated MRSA (HA-MRSA). HA-MRSA infectionstypically are associated with invasive procedures such as surgery or theuse of devices, such as intravenous tubing or artificial joints.

MRSA infections may also occur in the wider community, i.e. amonghealthy people; this form of MRSA infection, community-associated MRSA(CA-MRSA), often begins as a painful skin boil. It is spread byskin-to-skin contact and at-risk populations include groups thatfrequently are in contact with other people, such as high schoolwrestlers, child care workers and people who live in crowded conditions.

Many strains of MRSA have been identified and any strain of MRSA may betreated using the agents, compositions and methods of the invention.Particular strains are described below and identified in WO 2010/139957(incorporated herein by reference) and may be viewed as preferredstrains to be treated according to the present invention.

MRSA 1021 b is resistant to penicillin, clindamycin, gentamycin, fusidicacid erythromycin, trimethoprin, sulphamethoxazole, cefoxitin,ciprofloxacin, and fosphomycin glucose 6 phosphate.

MRSA 1141 b is resistant to penicillin, clindamycin, fusidic acid,erythromycin, trimethoprin, cefoxitin, ciprofloxacin and mupirocin.

MRSA 1108 is resistant to penicillin, fusidic acid, trimethoprin,cefoxitin, rifampicin and ciprofloxacin.

MRSA 1047 is resistant to penicillin, fusidic acid, erythromycin,trimethoprin, cefoxitin, ciprofloxacin, mupirocin, chloramphenicol, andfosphomycin glucose 6 phosphate.

MRSA 1040 is resistant to penicillin, gentamycin, fusidic acid,erythromycin, trimethoprin, sulphamethoxazole, tetracycline, cefoxitin,ciprofloxacin and mupirocin.

MRSA 1096 is resistant to penicillin, gentamycin, erythromycin,trimethoprin, sulphamethoxazole, cefoxitin and ciprofloxacin.

Staphylococcus aureus ATCC-43300 is resistant to methicillin andoxacillin.

Particularly preferred strains to be treated according to the presentinvention are MRSA 1040 and/or MRSA 1096.

In some embodiments, an MRSA infection may be treated using an agent orcomposition of the invention in combination with an antibiotic. In someembodiments, the antibiotic is a macrolide antibiotic, such asAzithromycin, Erythromycin, Clarithromycin, Telithromycin, Carbomycin A,Josamycin, Kitasamycin, Midecamycin/midecamycin acetate, Oleandomycin,Solithromycin, Spiramycin, Troleandomycin, Tylosin/tylocine orRoxithromycin. In some embodiments, the macrolide antibiotic isAzithromycin or Erythromycin.

In some embodiments the MDR bacterium is an Enterococcus faeciumbacterium. Alternatively viewed, the MDR bacterial infection is anEnterococcus faecium infection.

An example of an MDR strain of Enterococcus faecium is Enterococcusfaecium CCUG 37832 (TO-3).

Enterococcus faecium is commonly associated with endocarditis, urinarytract infections and infections in wounds. Enterococcus faecium may alsocause meningitis, e.g. neonatal meningitis. Thus, in some embodiments,the subject to be treated according to the methods and uses of theinvention has endocarditis, a urinary tract infection, an infected woundor meningitis, e.g. neonatal meningitis. In some embodiments, anEnterococcus faecium infection may be treated using an agent orcomposition of the invention in combination with an antibiotic. In someembodiments, the antibiotic is a DNA gyrase inhibitor, such as anaminocoumarin (e.g. novobiocin) or a quinolone (e.g. nalidixic acid orciprofloxacin). In some embodiments, the antibiotic is 2,4-Diamin, S.methizol, S. methoxa, S. dimetho, Sulfaceta, Trimethoprim, Flumeq,Levoflox, Pruliflox, Metronid or Nitrofur.

A bacterial infection to be treated or prevented by the agent orcomposition of the invention may be in any tissue or organ of thesubject to be treated, such as the lungs (including the respiratorytract), stomach, gastrointestinal tract (GIT), blood, skin (includingwounds, such as surgical wounds), bladder (including the urinary tract),kidney, ear, eye, meninges etc. Hence, the bacterial infection may be arespiratory infection, stomach infection, GIT infection, bloodinfection, skin infection, bladder infection, kidney infection, earinfection, eye infection, meningial infection etc. A skin infection mayinclude an infection of a mucosal membrane, such as the oral cavity,oesophagus or eye, e.g. cornea.

Thus, in some embodiments of the invention the infectious disease or adisease or condition exacerbated or caused by a bacterial infection mayinclude any one of bacterial pneumonia, cystic fibrosis, gastric ulcers,bacterial meningitis, Legionellosis (Legionnaires' disease),Legionellosis (Pontiac fever), Pertussis (Whooping cough),Salmonellosis, Tuberculosis, sepsis etc.

In some aspects of the invention the fungus may be a mold or yeast,preferably a yeast. The fungus may be selected from any one or more of aDermatophyte, Aspergillus sp. (such as Aspergillus fumigatus,Aspergillus nigricans or flavescens), Zygomycota sp., Fusarium sp.,Trichophyton sp., Basidiobolus ranarum, Piedraia sp. (such as Piedraiahortae), Blastomyces dermatitidis, Candida sp. (such as Candidaalbicans), Chrysosporium, Coccidioides sp. (such as Coccidioides immitisand Coccidioides posadasii), Conidiobolus sp. (such as Conidioboluscoronatus and Conidiobolus incongruus), Cryptococcus sp. (such asCryptococcus gattii and Cryptococcus neoformans), Histoplasma sp. (suchas Histoplasma farciminosum and Histoplasma capsulatum), Exserohilumrostratum, Cladosporium sp., Saccharomyces sp., Lacazia loboi,Paracoccidioides brasiliensis, Penicillium marneffei, Pneumocystisjirovecii, Sporothrix schenckii, Diheterospora zeaspora, Absidiacorymbifera, Apophysomyces elegans, Mucor indicus, Rhizomucor pusillus,Rhizopus oryzae, Cunninghamella bertholletiae, Cokeromyces recurvatus,Saksenaea vasiformis, Syncephalastrum racemosum, Conidiobolus sp. (suchas Conidiobolus coronatus and Conidiobolus incongruus).

By way of a representative example, fungi that cause infections orinfectious diseases that may be treated or prevented by the agents andcompositions described herein include fungi from the genera Candida,Aspergillus, Pneumocystis, Penicillium and Fusarium. Representativefungal species include, but are not limited to, Candida albicans,Candida dubliniensis, Cryptococcus neoformans, Histoplama capsulatum,Aspergillus fumigatus, Coccidiodes immitis, Paracoccidioidesbrasiliensis, Blastomyces dermitidis, Pneomocystis camii, Penicilliummameffi and Alternaria alternate. etc.

A variety of environmental and physiological conditions can contributeto the development of fungal diseases and infections. Fungal infections(mycoses) commonly start in the lungs or on the skin, e.g. a fungalinfection (a mycosis) may result from the inhalation of fungal spores orlocalized colonization of the skin may initiate persistent infections.Thus, a fungal infection (mycosis) to be treated or prevented by theagent or composition of the invention may be in any tissue or organ ofthe subject to be treated, such as the lungs (including the respiratorytract), skin (including wounds), mouth, ear, eye etc. Hence, the fungalinfection may be a respiratory infection, skin infection, ear infection,eye infection etc. Thus, in some embodiments of the invention theinfectious disease or a disease or condition exacerbated or caused by afungal infection may include any one of Allergic bronchopulmonaryaspergillosis, Aspergilloma, Aspergillosis, Athlete's foot,Basidiobolomycosis, Black piedra, Blastomycosis, Candidiasis, Chronicpulmonary aspergillosis, Coccidioidomycosis, Conidiobolomycosis,Cryptococcosis, Dermatophytosis, Endothrix, Epizootic lymphangitis,Esophageal candidiasis, Exothrix, Fungal meningitis, Fungemia,Histoplasmosis, Lobomycosis, Myringomycosis, Oral candidiasis,Paracoccidioidomycosis, Penicilliosis, Piedra, Pneumocystis pneumonia,Sporotrichosis, Tinea such as Tinea barbae, Tinea capitis, Tineacorporis, Tinea cruris, Tinea faciei, Tinea incognito, Tinea nigra,Tinea versicolor, Zeaspora, Zygomycosis, etc.

As noted above, in some embodiments the agent or composition as definedherein is used in combination with one or more additional active agents,e.g. a cytotoxic or cytostatic compound, in order to enhance orcomplement the effect of agent or composition defined herein. In someembodiments, the additional active agent may be used to treat symptomsof the microbial infection or infectious disease, e.g. secondarysymptoms, such agents may be, e.g. an anti-inflammatory compound,steroid (e.g. a corticosteroid) etc. and will be dependent on the natureof the disease, including the severity of the symptoms etc. However, insome embodiments, the agent as defined herein may be used alone, i.e. asthe only active agent in a composition and/or medicament.

In some embodiments, e.g. where the agent or composition is used totreat or prevent a fungal infection or infectious disease, theadditional active agent is a cytostatic or cytotoxic agent. Inparticularly preferred embodiments, the cytotoxic or cytostatic agent isan intracelluarly-active agent, i.e. it targets an intracellularprocess. In other words, in some embodiments, the cytotoxic orcytostatic agent does not act at or on the cell wall or cell membrane,i.e. it does not exert its cytotoxic or cytostatic effect by affectingthe cell wall or cell membrane, e.g. by inhibiting cell wall synthesisor by permabilizing the cell.

By “cytostatic agent” is meant an agent which is capable of inhibitingor suppressing the growth and/or multiplication(replication/proliferation) of microbial cells, e.g. an anti-microbialagent, such as an antibiotic or antifungal (an antimycotic) agent.

Included as cytostatic agents are cytotoxic agents and any agent whichmay be indicated for an antimicrobial application. Thus, included areagents used in antimicrobial treatment protocols. However, cytostaticagents that are not typically used for suppressing or inhibitingmicrobial growth may find utility in combination with the agents definedherein, e.g. agents that generally have an effect on cell growth, suchas chemotherapeutic agents, particularly DNA damaging agents.

In some embodiments, the cytostatic or cytotoxic agent is an antibioticand/or antifungal agent.

Suitable antibiotic agents include but are not limited to any one ormore of Aminocoumarins (such as Novobiocin, Albamycin, Coumermycin andClorobiocin), Aminoglycosides (such as Amikacin, Apramycin, Gentamicin,Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin andSpectinomycin), Ansamycins (such as Geldanamycin, Herbimycin, Rifaximinand Streptomycin), Carbapenems (such as Ertapenem, Doripenem, Cilastatin(‘Imipenem’) and Meropenem), Cephalosporins (such as Cefadroxil,Cefazolin, Cefalothin (‘Cefalotin’), Cefalexin, Cefaclor, Cefamandole,Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefoperazone,Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime,Ceftriaxone, Cefepime, Ceftaroline fosamil and Ceftobiprole)Glycopeptides (such as Teicoplanin, Vancomycin and Telavancin),Lincosamides (such as Clindamycin and Lincomycin), Lipopeptides (such asDaptomycin), Macrolides (such as Azithromycin, Clarithromycin,Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin,Telithromycin and Spiramycin), Monobactams (such as Aztreonam),Nitrofurans (such as Furazolidone and Nitrofurantoin), Oxazolidonones(such as Linezolid, Posizolid, Radezolid and Torezolid), Penicillins(such as Amoxicillin, Ampicillin, Azlocillin, Carbenicillin,Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin,Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin,Temocillin and Ticarcillin), Penicillin combinations (such asAmoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactamand Ticarcillin/clavulanate), Polyethers (such as Monensin),Polypeptides (such as Bacitracin, Colistin and Polymyxin B), Quinolones(such as Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,Trovafloxacin, Grepafloxacin, Sparfloxacin and Temafloxacin);Sulfonamides (such as Mafenide, Sulfacetamide, Sulfadiazine, Silversulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole,Sulfanilimide, Sulfasalazine, Sulfisoxazole, Sulfamethoxazole(Co-trimoxazole, TMP-SMX, ‘Trimethoprim’) and Sulfonamidochrysoidine),Tetracyclines (such as Demeclocycline, Doxycycline, Minocycline,Oxytetracycline and Tetracycline) and Others (such as Clofazimine,Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid,Pyrazinamide, Rifampicin (‘Rifampin’), Rifabutin, Rifapentine,Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid,Metronidazole, Mupirocin, Platensimycin, Quinupristin (Dalfopristin),Thiamphenicol, Tigecycline, Tinidazole and Trimethoprim).

Suitable antifungal compounds (antimycotics) include but are not limitedto any one or more of Polyene antifungals (such as Amphotericin B,Candicidin, Filipin, Hamycin, Natamycin, Nystatin and Rimocidin),Imidazoles (such as Bifonazole, Butoconazole, Clotrimazole, Econazole,Fenticonazole, Isoconazole, Ketoconazole, Miconazole, Omoconazole,Oxiconazole, Sertaconazole, Sulconazole and Tioconazole), Triazoles(such as Albaconazole, Fluconazole, Isavuconazole, Itraconazole,Posaconazole, Ravuconazole, Terconazole and Voriconazole), Thiazoles(such as Abafungin), Allylamines (such as Amorolfin, Butenafine,Naftifine and Terbinafine), Echinocandins (such as Anidulafungin,Caspofungin and Micafungin) and Others such as Benzoic acid, Ciclopirox,Flucytosine or 5-fluorocytosine, Griseofulvin, Haloprogin, Polygodial,Tolnaftate, Undecylenic acid and Crystal violet.

Other cytostatic agents that may find utility in the invention may begrouped into different classes according to their mechanism of actionand all of these classes are contemplated herein. Thus, the cytostaticagent may be an alkylating agent, a cross-linking agent, anintercalating agent, a nucleotide analogue, an inhibitor of spindleformation, and/or an inhibitor of topoisomerase I and/or II. Other typesor classes of agent include anti-metabolites, plant alkaloids andterpenoids, or an anti-tumour antibiotic. Preferably, it is analkylating agent.

Alkylating agents modify DNA by alkylating nucleosides, which leads tothe prevention of correct DNA replication. Nucleotide analogues becomeincorporated into DNA during replication and inhibit DNA synthesis.Inhibitors of spindle formation disturb spindle formation, leading tothe arrest of mitosis during metaphase. Intercalating agents intercalatebetween DNA bases, thereby inhibiting DNA synthesis. Inhibitors oftopoisomerase I or II affect the torsion of DNA, thereby interferingwith DNA replication.

Suitable cytostatic agents are known in the art, but by way of exampleMMS (Methyl methanesulphonate), actinomycin D, BCNU (carmustine),carboplatin, CCNU, Campothecin (CPT), cantharidin, Cisplatin,cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel,Doxorubicin, DTIC, epirubicin, Etoposide, gefinitib, gemcitabine,ifosfamide and/or irinotecan, ionomycin, Melphalan, Methotrexate,Mitomycin C (MMC), mitozantronemercaptopurine, Oxaliplatin, Paclitaxel(taxol), PARP-1 inhibitor, taxotere, temozolomide (TZM), teniposide,topotecane, treosulfane vinorelbine, vincristine, vinblastine,5-Azacytidine, 5,6-Dihydro-5-azacytidine and 5-fluorouracil are namedherein.

The skilled person will be aware of suitable dosage ranges for any givencytostatic agent and, in one embodiment, the cytostatic agent is presentin the pharmaceutical composition, or administered to the subject, inits typical dose range. In an advantageous embodiment, a lower dose ofthe cytostatic agent may be used because the agent defined hereinsensitises the microbial cells to the cytostatic agents and so when usedin combination with the agent of the invention, a lower dose of thecytostatic agent will have the same or a comparable therapeutic effectas a higher dose of the cytostatic agent on its own.

As discussed above, a suitable dose for the agent or composition asdefined herein may be defined as a dose that is sufficient, either aloneor in combination with an additional active agent, to inhibit microbialcell growth (e.g. in vivo or in vitro). In some embodiments, a suitabledose may be defined as a dose that is sufficient, either alone or incombination with an additional active agent to kill the majority of themicrobial cells causing, or associated with, the infection or infectiousdisease. In some embodiments, a suitable dose for the agent orcomposition as defined herein may be defined as a dose that issufficient to sensitize a microbe to a cytotoxic or cytostatic agent,wherein contacting the microbe with (e.g. treatment with oradministration of) an agent and a cytotoxic or cytostatic agent issufficient to inhibit microbial cell growth (e.g. in vivo or in vitro)and/or sufficient to kill the majority of the microbial cells causing,or associated with, the infection or infectious disease.

In some embodiments, a does may be defined as a dose that does notinduce apoptosis in animal cells significantly, i.e. an apoptosisnon-inducing dose for animal cells, particularly human cells. Thus, asuitable dose may be defined as a “low dose” or “low amount” of theagent (e.g. oligopeptidic compound), which may be seen as a dose oramount that is not sufficient to cause or induce apoptosis in animalcells either directly or indirectly.

The “majority of cells” may be defined as at least 50% of the microbialcells, e.g. at least 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% of the cells, e.g. cells causing, or associated with, theinfection or infectious disease.

Thus a dose that does not induce apoptosis in animal cells significantlymay be viewed as a dose that causes or induces apoptosis of less than20% of the animal cells in the target area, e.g. less than 15, 10, 9, 8,7, 6, 5, 4, 3, 2 or 1% of the animal cells in the target area. Thetarget area may be the area to which the agent or composition isadministered, e.g. an organ or a portion thereof.

The effective dose or amount of agent may depend on the characteristicsof the peptide, e.g. the strength of the interaction between the PCNAinteracting motif and the binding domain of the target protein(s).Furthermore, effective dose or amount of the agent may depend upon thenature of the compound used (i.e. peptide, nucleic acid molecule etc),the mode of administration, the course of treatment, the age and weightof the patient, the medical indication, the body or body area to betreated, or the in vitro use, and may be varied or adjusted according tochoice. Generally however, a low dose or amount may result in an activeconcentration range of about 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0,1.25, 1.50, 1.75, 2.0, 3.0, 4.0, 5.0 to 10 μM, e.g. 0.01 to 10 μM, e.g.0.05 to 7.5 μM, such as 0.1 to 7.5 μM, e.g. 0.5 to 5 μM. A high dose oramount may result in an active concentration range of about 1.0, 2.0,3.0, 4.0, 5.0, 7.5, 10, 15, 20, 25, 30, 40 to 50 μM, e.g. 1.0 to 50 μM,e.g. 2.0 to 40 μM, such as 3.0 to 30 μM, e.g. 5.0 to 25 μM. Saidconcentrations are determined by reference to the amount of the compounditself and thus appropriate allowances should be made to take intoaccount the purity of the composition.

The subject is an animal (i.e. any human or non-human animal),preferably a mammal, most preferably a human.

As noted above, the agent or composition as defined herein may beprovided or administered via a product, device, implant or material towhich the agent or composition has been applied, impregnated orchemically bonded. Hence, the invention also provides a product,material, device or implant which is coated, impregnated or chemicallybonded with an agent or composition as described herein. The inventionalso extends to the use of such products, materials, devices or implantsin the methods and uses as described herein. In particular, theproducts, materials, devices or implants may be coated, impregnated orchemically bonded with an agent or composition as described herein toprevent or inhibit the formation of a biofilm.

Thus, the present invention also provides a method of preventing orinhibiting the formation of a bacterial biofilm on a product, material,device or implant, said method comprising:

(i) providing a product, material, device or implant; and

(ii) coating or impregnating said device with an agent or composition asdefined herein, or chemically bonding an agent or composition as definedherein to said product, material, device or implant.

To this end, bandages, plasters (e.g. adhesive patches), gauze, surgicaltape, cotton swabs or other absorbent materials, e.g. a puff, fleece, orsponge, supportive matrices or wound dressings may be coated,impregnated or chemically bonded with an agent or composition asdescribed herein. For example, many compositions can be applied to theskin using dermal patches that are well described in the art, e.g. US2008/0038300, US 2009/0043236, WO 2005/067499 and WO 2009/085302, whichare incorporated herein by reference. In some embodiments, the materialcomprising the agent or composition as described herein may be in theform of a device that can be, e.g. worn by the subject to be treated.For instance, the agent or composition as described herein may beapplied, impregnated or chemically bonded onto a material or supportivematrix that forms all or part of a diaper, glove, sock etc.

In some embodiments, the product or material is a bandage, plaster (e.g.adhesive patch), gauze, surgical tape, cotton swab, puff, fleece,sponge, supportive matrix, wound dressing, diaper, glove or sock.

In still further embodiments, the device or implant may be a medical orsurgical device or implant. For instance, the device or implant may beselected from, but is not limited to, a stent (e.g. coronary stent), eartube (tympanostomy tube), artificial eye lens (i.e. a pseudophakos orintra-ocular lens), an orthopedic implant (e.g. screw, pin, plate orrod, such as for traumatic fracture repair or spinal fusion), anartificial bone (e.g. a spinal disc, hip, knee etc), a dental implant(e.g. an artificial tooth or part thereof), a cardiac device (e.g. animplantable cardioverter defibrillator, pacemaker etc.), a cosmeticimplant (e.g. breast implant), intra-uterine device (IUD), a catheter(e.g. central venous or urinary catheter) or a prosthetic device.

In some embodiments, the product or device may be a contact lens orcontact lens storage case.

The agent, composition, product, material, device or implant can beincluded in a container, pack, or dispenser together with instructionsfor administration and/or use.

The invention will now be further described with reference to thefollowing non-limiting Examples and Figures in which:

FIG. 1 shows graphs that demonstrate that ATX-101 (SEQ ID NO:1198, whichcontains the APIM motif, RWLVK (SEQ ID NO: 28)) increases thesensitivity of yeast (Saccharomyces cerevisiae) to a cytostatic agent(cisplatin), wherein: (A) shows the growth of yeast in the presence ofvarious concentrations of cisplatin; (B) shows the growth of yeast inthe presence of various concentrations of ATX-101 (APIM); (C) shows thegrowth of yeast in the presence of various concentrations of ATX-101 incombination with 500 μM cisplatin; and (D) shows the growth of yeast inthe presence of various concentrations of ATX-101 in combination with125 μM cisplatin.

FIG. 2 shows graphs that demonstrate that Hog1 mutant yeast cells haveincreased sensitivity to ATX-101 in combination with cisplatin relativeto wild-type yeast cells, wherein: (A) shows the growth of wild-typeyeast in the presence of various concentrations of ATX-101 incombination with 125 μM cisplatin; and (B) shows the growth of Hog1yeast in the presence of various concentrations of ATX-101 incombination with 125 μM cisplatin.

FIG. 3 shows a laser scanning microscope image of yeast cells incubatedwith ATX-101 labelled with FAM.

FIG. 4 shows a graph that demonstrates that the growth of gram-negativebacteria, Pseudomonas aeruginosa ATCC 15692, Acinetobacter baumanni ATCC19606, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa TO-5A(clinical isolate) is inhibited by varying concentrations of APIMpeptide, ATX-101. Growth of the strains is given relative to growth incultures without ATX-101.

FIG. 5 shows a graph that demonstrates that the growth of gram-positivebacteria, Enterococcus faecium CCUG 37832, Enterococcus faecium CTC 492and Micrococcus luteus ATCC 9341, is inhibited by varying concentrationsof APIM peptide, ATX-101.

FIG. 6 shows a graph that demonstrates that the growth of thegram-positive bacterium Enterococcus faecium CTC 492 is more sensitiveto: (A) Ampicillin; and (B) Novobiocin, (both cytotoxic agents, i.e.antibiotics) when grown in the presence of ATX-101.

FIG. 7 shows a graph that demonstrates that the growth of thegram-negative bacterium Escherichia coli NCIMB 12210 is more sensitiveto Novobiocin (a cytotoxic agent, i.e. an antibiotic) when grown in thepresence of ATX-101.

FIG. 8 shows a graph showing the results of FRET analysis. NormalisedFRET (N_(FRET)) measurements are shown between EYFP (yellow fluorescentprotein)/ECFP (cyan fluorescent protein) (Lane 1, background due todimerisation of the tags). EYFP-APIM motif/ECFP-PCNA for various motifsare shown in the other lanes.

FIG. 9 shows a graph that demonstrates that the growth of the MDRbacterium Methicillin-resistant Staphylococcus aureus (MRSA 1040) ismore sensitive to macrolide antibiotics, azithromycin and erythromycin,when grown in the presence of ATX-101.

FIG. 10 shows a graph that demonstrates that various APIM peptidevariants are capable of reducing the MIC of erythromycin required toinhibit the growth of the MDR bacterium Methicillin-resistantStaphylococcus aureus (MRSA 1040), wherein: ATX-101-STD is SEQ ID NO:1198; ATX-101-Sv is SEQ ID NO: 1203; ATX-101-Av is SEQ ID NO: 1204;ATX-101-Ls is SEQ ID NO: 1211; ATX-101-P is SEQ ID NO: 1212; andATX-101-LT is SEQ ID NO: 1208.

FIG. 11 shows graphs that demonstrate that the growth of the MDRbacterium E. faecium CCUG 37832 (TO-3) is more sensitive to various DNAgyrase inhibitors, when grown in the presence of ATX-101.

FIG. 12 shows (A) a graph showing the growth of E. coli in which theoverexpression of various proteins or peptides is induced or not inducedand demonstrates that the induction of an APIM peptide (SEQ ID NO: 28),alone or as part of a fusion protein with EYFP, inhibits bacterialgrowth. (B) shows a Western blot showing levels of EYFP-proteins inun-induced (left) versus induced (right) cell cultures.

FIG. 13 shows graphs showing the growth of E. coli in which theoverexpression of various APIM peptides is induced (I) or not induced(NI). The APIM sequences are: (A) SEQ ID NO: 28; (B) SEQ ID NOs: 1206,1207 and 76; (C) SEQ ID NOs: 41, 40 and 52; (D) SEQ ID NO: 58.

FIG. 14 shows graphs showing the growth of E. coli in the presence of 30μM of various APIM peptides. The same data is presented in (A) and (B),however the control growth curves are not shown in (B). ATX-101 is SEQID NO: 1198; ATX-101-P is SEQ ID NO: 1212; FSL is SEQ ID NO: 1206; FLSis SEQ ID NO: 1210; SV is SEQ ID NO: 1203; AV is SEQ ID NO: 1204; LT isSEQ ID NO: 1208; LS is SEQ ID NO: 1211; R11 is SEQ ID NO: 337; Neg contris no peptide added.

FIG. 15 shows graphs showing the growth of E. coli in the presence of 30μM of various APIM peptides after the bacteria have been irradiated withUVC. The same data is presented in (A) and (B), however the controlgrowth curves are not shown in (B). ATX-101 is SEQ ID NO: 1198;ATX-101-P is SEQ ID NO: 1212; FSL is SEQ ID NO: 1206; FLS is SEQ ID NO:1210; SV is SEQ ID NO: 1203; AV is SEQ ID NO: 1204; LT is SEQ ID NO:1208; LS is SEQ ID NO: 1211; R11 is SEQ ID NO: 337; Neg contr is nopeptide added.

FIG. 16 shows graphs showing the growth of E. coli in the presence ofATX-101 (SEQ ID NO: 1198) at various concentrations, without UVCirradiation (left panels) or with UVC irradiation (right panels). R11 isSEQ ID NO: 337 and Neg contr is no peptide added. (A) 15 μM, (B) 7.5 μMand (C) 3.75 μM.

FIG. 17 shows micrographs of biofilm formation of the MDR bacteriumMethicillin-resistant Staphylococcus aureus (MRSA 1040) in the presenceof 7 μg/ml ATX-101 (SEQ ID NO: 1198) (left panels) or no peptide (rightpanels) over a period of 36 hours and demonstrates that biofilmformation is inhibited by ATX-101.

FIG. 18 shows micrographs of biofilm formation of the MDR bacteriumMethicillin-resistant Staphylococcus aureus (MRSA 1040) in the presenceof 0.8 μg/ml ATX-101 (SEQ ID NO: 1198) (left panels) or no peptide(right panels) over a period of 36 hours and demonstrates that biofilmformation is inhibited by ATX-101.

EXAMPLES

The inventors have surprisingly found that an oligopeptidic compoundcomprising an APIM motif and a cell-penetrating peptide is imported intomicrobial cells and is capable of inhibiting the growth of said cells.It is thought that oligopeptidic compounds comprising an APIM motif maycompete with PCNA-like proteins in microorganisms for proteins thatinteract with said PCNA-like proteins, thereby inhibiting variouscellular processes, e.g. DNA synthesis, signal transduction etc. Theeffects of oligopeptidic compounds comprising an APIM motif on microbialcell have been established in both bacteria and fungi using an exemplarycell-penetrating APIM-containing peptide ATX-101 (SEQ ID NO:1198, whichcontains the APIM motif, RWLVK (SEQ ID NO: 28)).

The data presented below suggest that oligopeptidic compounds comprisingan APIM motif are useful as antimicrobial agents, e.g. antibiotics andantimycotics, either alone or in combination with other cytostatic orcytotoxic agents. Accordingly, the data supports the use ofoligopeptidic compounds comprising an APIM motif in the treatment orprevention of microbial infections or microbial infectious diseases.

Example 1 Determining the Effect of ATX-101 on Yeast (Saccharomycescerevisiae

The homozygote diploid mutant library of yeast strains was purchasedfrom EUROSCARF, Institute of Microbiology, University of Frankfurt.Growth studies on wild-type (wt) yeast and the Hog1 mutant wereperformed. Microplates (Greiner 655163) containing 120 μl 2×MES and 1.5×N-base medium per well were inoculated with 20 μl (per well) of eachyeast strain (from frozen stock cultures) and a reference strain (wildtype BY4743). The microplate cultures were grown over night (ON) (30°C., 900 rpm at 3 mm amplitude, 85% humidity), ensuring growth well intothe stationary growth phase in order to reduce variation caused bydifferences in growth rates. At day 2, 10 μl was transferred from theON-cultures to 200 μl 2×MES 1.5× N-base per well in 96-well microplates,mixed (900 rpm at 3 mm amplitude, 30 sec) and grown to OD 0.15-0.2before addition of different doses of cisplatin and/or ATX-101. The OD(600 nm) in each well was read every 20 minutes using an integratedBeckman Coulter Paradigm microplate reader. The growth of the yeaststrains in the microplates was monitored for approximately 25 hours.

FIG. 1 shows that cisplatin at a concentration of 125 μM is notsufficient to retard growth of the yeast cells (FIG. 1A). A cisplatinconcentration of 500 μM is required to retard growth and even at 2000μM, growth is not completely inhibited. Similarly, ATX-101 (labeled asAPIM) at a concentration of 10 μM does not retard growth and aconcentration of 20 μM or more is necessary to retard growth of theyeast cells (FIG. 1B). However, the combination of cisplatin at 500 μMand ATX-101 at 20 μM is capable of inhibiting growth of the yeast cellsafter approximately 7 hours (FIG. 10). Thus, ATX-101 (an APIM peptide)and cisplatin (a cytostatic agent) have a synergistic effect on yeast,when combined. In fact, the effect is seen when cisplatin is used at aconcentration that, alone, has no effect on yeast growth, 125 μM (FIG.1D). It is also evident from this experiment that high concentrations ofATX-101 alone will be capable to inhibiting growth of yeast cells.

FIG. 2 shows that Hog1 yeast mutants (FIG. 2B) show increasedsensitivity to ATX-101 in combination with cisplatin. A combination of10 μM ATX-101 and 125 μM cisplatin is sufficient to retard growth andhigher concentrations of ATX-101 result in complete inhibition of cellgrowth. Hog1 is a protein kinase in the a mitogen activated protein(MAP) kinase pathway in yeast, which is required for adaptation toosmotic stress. Thus, these data suggest that ATX-101 may be acting byinterfering with intracellular signalling in addition to DNA synthesisand repair mechanisms.

Based on these results, APIM peptides are expected to be useful asantimycotic agents for all fungi.

Example 2 Cellular Import of ATX-101

Yeast cells (Saccharomyces cerevisiae) were grown in LB media. Afluorescently labelled APIM peptide (ATX-101-FAM) was added to the mediaand incubated for 1-2 minutes before an aliquot of cells was removed andanalyzed on a Zeiss LSM 510 Meta laser scanning microscope equipped witha Plan-Apochromate 63×/1.4 oil immersion objective.

FIG. 3 shows that ATX-101 is imported into yeast cells, therebydemonstrating that it must be acting intracellularly, i.e. unlike manyantimicrobial compounds ATX-101 is not antimicrobial due to its effectson cell walls or membranes, i.e. it does not function by permeabilizingcells.

Example 3 Determining the Effect of ATX-101 on Bacteria

The minimum inhibitory concentration (MIC) of ATX-101 was determined forvarious gram negative and gram positive bacteria. The bacteria,Pseudomonas aeruginosa ATCC 15692, Acinetobacter baumanni ATCC 19606,Escherichia coli ATCC 25922, Pseudomonas aeruginosa TO-5A (clinicalisolate), Enterococcus faecium CCUG 37832, Enterococcus faecium CTC 492,Micrococcus luteus ATCC 9341 and Escherichia coli NCIMB 12210 were grownin the presence of various concentrations of ATX-101 and/or antibiotic.

Robotic MIC Assay:

ATX-101 was dissolved in Mueller-Hinton broth to 1.25 times of thedesired assay concentration. Antibiotics were dissolved inMueller-Hinton broth and Mueller-Hinton broth with ATX-101 at aconcentration of 1.25 times the highest desired assay concentrations.Antibiotics were pharmaceutical grade purchased from Sigma-Aldrich.

Two-fold serial dilutions of antibiotics were made in Mueller-Hintonwith different concentrations of ATX-101, and the solutions were placedin four parallel wells in Nunc 384-well micro plates (30 μl per well inNunc 242757 microplates). A group of 8 wells with no addition ofantibiotics for each ATX-101 concentration was included on each microplate as a growth reference.

At the day of analysis, overnight TSB cultures inoculated from freezestocks (6 ml in 50 ml tube tilted to 45-degrees angle, 200 rpm, 2.5 cmamplitude, 37° C.) were diluted in TSB until the OD600 was 0.10, andfurther diluted 1:40 in Mueller-Hinton broth. Each well in the 384-wellassay plates was inoculated with 7.5 μl of the diluted culture, givingthe same dilution of the culture in the assay cultures. The microplateswere placed in plastic bags and incubated without shaking at 37° C. Theoptical density at 600 nm in the microwells was measured afterapproximately 19 hours of incubation, and the relative growth yield ineach well was calculated based on the growth in the reference groups.The MIC value was set to the highest concentration giving less than 30%growth in all 4 parallel wells within the sample groups. The microplateswere further incubated for 6 hours, and optical density in the cultureswas measured once more for confirmation of the estimated MIC-values.

FIG. 4 shows that the growth of various gram negative bacteria isinhibited by ATX-101 from about 1 μM. FIG. 5 shows that the growth ofvarious gram positive bacteria is inhibited by ATX-101 from about 1 μM.The MIC for the gram negative bacteria tested to date was determined tobe in the range of 5-10 μg/ml. The MIC for gram positive bacteria testedto date was determined to be in the range of 1 μg/ml. These data suggestthat, in general, gram positive bacteria are more sensitive to APIMpeptides than gram negative bacteria. However, based on these results,APIM peptides are expected to be useful as antibiotic agents for allbacteria.

FIG. 6 shows that Enterococcus faecium CTC 492 shows increasedsensitivity to ampicillin and novobiocin in combination with ATX-101. Acombination of 6.5 μg/ml or 13 μg/ml ATX-101 is sufficient to inhibitbacterial growth at a concentration of about 0.5 μg/ml ampicillin,whereas a concentration of 1 μg/ml ampicillin without ATX-101 wasrequired to achieve the same level of inhibition. Similarly, 6.5 μg/mlor 13 μg/ml ATX-101 enhances the inhibition of bacterial growth at aconcentration of about 0.5-2 μg/ml novobiocin. FIG. 7 shows that thatATX-101 is capable of reducing the MIC for novobiocin by 16-fold. Thus,these data suggest that ATX-101 has an additive or synergisticinhibitory effect on bacterial growth in combination with an antibiotic.FIG. 6 also demonstrates that higher concentrations of ATX-101, e.g. 20μg/ml, are sufficient to kill bacteria, as no growth was observed atthis concentration, with or without ampicillin or novobiocin.

Based on these results, APIM peptides are expected to be useful incombination with other antibiotic agents, i.e. may enable knownantibiotics to be used effectively at lower concentrations.

Example 4 Determining the Effect of Other APIM Peptides on Bacteria

The inventors have shown that other APIM peptides, i.e. comprising PCNAbinding motifs that are different to the motif in ATX-101, are alsoeffective antibiotics. In this respect, peptides comprising thesequences: MDRWSVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1203) andMDRWAVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1204), i.e. wherein X₃ and X₄ areSV and AV, respectively, are particularly effective and show lower MICsthan ATX-101. These data indicate that the antimicrobial effect arisesfrom the PCNA interacting motif, because the other domains in thesepeptides are identical.

Example 5 In Silico Characterisation of APIM Consensus Motif

The inventors have performed sequence analyses to determine how muchvariation within the APIM motif occurs naturally, i.e. in nativesequences across a number of species. As PCNA is highly conserved acrosseukaryotic organisms, it is expected that sequence variation of the APIMmotif in orthologues of polypeptides that are thought to interact withPCNA is representative of the variation that may be used in theoligopeptidic compounds of the invention, i.e. variation of amino acidswithin the APIM motif at some positions, particularly X₃ and X₄, may bepermitted without losing affinity to PCNA.

The inventors used identified 657 human polypeptide sequences thatcomprise the motif [K/R]-[F/W/Y]-[A/L/V/I]-[A/L/V/I]-[K/R] (SEQ IDNO:19) from a possible 21,673 polypeptide sequences. Of the 657sequences identified, 291 were excluded because insignificantinformation about the function of the polypeptides was available. Theremaining 366 were considered to be polypeptides that are likely tointeract with PCNA and these sequences were used to identify orthologuesin: Bos taurus (288 orthologues); Rattus norvegicus (286 orthologues);Mus musculus (312 orthologues); Gallus gallus (236 orthologues); Xenopustropicalis (200 orthologues); Danio rerio (189 orthologues);Caenrhabditis elegans (102 orthologues); Drosophila melanogaster (136orthologues); and Saccharomyces cerevisiae (65 orthologues). Alignmentof the domains of the orthologues that comprise the APIM motif suggestedthat the motif may defined as:

[R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T/N/Q/C]-[L/I/V/A/M/G/S/T/N/Q/R/H/K/C]-[K/R/H/P](SEQ ID NO: 2), wherein specific combinations of amino acids atpositions 3 and 4 that were identified in the orthologues include:

LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA, AI, AM, LM, LS,LT, IS, MV, TV, AA, IM, LN, LQ, VM, TL, SL, IT, VT, LG, MA, ML, NL, QL,QI, TI, SI, AS, VS, SV, CA, IG, LR, VR, TK and IR. Particularly commoncombinations are LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA,AI, AM, LM, LS and LT, the most common being LL, LA, LV, AL, VL, VI, LI,IL, VV, VA, IV, II, AV, IA and AI.

Thus, the broadest definition of the APIM motif was derived from thisanalysis, and all polypeptides comprising an APIM motif according tothis definition could reasonably be expected to interact with, i.e. bindto, PCNA.

Example 6 In Vivo Characterisation of APIM Consensus Motif

This work described in this Example investigates interaction betweenAPIM peptides and PCNA.

In living S-phase cells, PCNA tagged with green fluorescent protein(EGFP) forms distinct foci representing sites of replication and thuscan be used as a S-phase marker.

PCNA tagged with cyan fluorescent protein (ECFP) was co-expressed withvarious APIM peptide constructs fused with yellow fluorescent protein(EYFP). To examine the degree of proximity of APIM peptides and PCNA,fluorescence resonance energy transfer (FRET) was measured.

Live HeLa cells were examined 16-24 hours after transient transfection(by Fugene 6 (Roche Inc.) according to the manufacturer'srecommendations) of ECFP and EYFP fusion constructs. Fluorescent imageswere acquired using a Zeiss LSM 510 Meta laser scanning microscopeequipped with a Plan-Apochromate 63×/1.4 oil immersion objective.Enhanced cyan fluorescent protein (ECFP) was excited at λ=458 nm anddetected at λ=470-500 nm and enhanced yellow fluorescent protein (EYFP)was excited at λ=514 nm and detected at λ=530-600 nm, using consecutivescans. The thickness of the slice was 1 μm.

Fluorescent resonance energy transfer (FRET) occurs if the tags (EYFPand ECFP) are less than 100 Å (10 nm) apart. We detected FRET using thesensitised emission method, measuring acceptor (EYFP) emission upondonor (ECFP) excitation. We had FRET when the intensity of emitted lightfrom EYFP after excitation of the ECFP fluorochrome was stronger thanthe light emitted by ECFP or EYFP-tagged proteins alone, afterexcitation with the EYFP and ECFP lasers respectively (bleed through),given by the equation: FRET=I₂−I₁ (I_(D2)/I_(D1))−I₃ (I_(A2)/I_(A3))is >0. FRET was normalised for expression levels using the equation:N_(FRET)=FRET/(I₁×I₃)^(1/2). N_(FRET) was calculated from meanintensities (I) within a region of interest (ROI) containing more than25 pixels where all pixels had intensities below 250 and the averageintensities were between 100 and 200 for both the donor and the acceptorconstructs. Channel 1 (ECFP) and 3 (EYFP) were measured as describedabove for imaging, and channel 2 (FRET) was excited with λ=458 nm anddetected at λ=530-600 nm. I_(D1, D2, D3) and I_(A1, A2, A3) weredetermined for cells transfected with ECFP and EYFP constructs only,with same settings and same fluorescence intensities as co-transfectedcells (I₁ and I₃). ECFP-PCNA and EYFP-PCNA were included as positivecontrols, and due to dimerisation of co-expressed tags, ECFP and EYFPproteins expressed from empty vectors were included as negative controlsin all experiments.

FIG. 8 shows that a significant FRET signal could be detected for all ofthe variants tested, which verifies that a variety of peptides withinthe APIM motif definition described herein (and that occur inpolypeptides that are expected to interact with PCNA) are capable ofinteracting with PCNA and would therefore be expected to find utility inthe method and uses described herein, i.e. as anti-microbial peptides.

Example 7 Determining the Effect of APIM Peptides on the MinimumInhibitory Concentration of Various Antibiotics on Bacteria

The APIM peptide ATX-101 was used to determine the effect of APIMpeptides on the MIC for various antibiotics on a range of bacteria.

The APIM peptide was dissolved in Mueller-Hinton broth to 1.25 times ofthe desired assay concentration. Antibiotics were dissolved inMueller-Hinton broth and Mueller-Hinton broth with APIM at aconcentration of 1.25 times the highest desired assay concentrations.Antibiotics were pharmaceutical grade purchased from Sigma-Aldrich.

Two-fold serial dilutions of antibiotics were made in Mueller-Hintonwith different concentrations of APIM, and the solutions were placed infour parallel wells in Nunc 384-well micro plates (30 μl per well inNunc 242757 microplates). A group of 8 wells with no addition ofantibiotics for each APIM concentration was included on each micro plateas growth reference.

At the day of analysis, overnight TSB cultures inoculated from freezestocks (6 ml in 50 ml tube tilted to 45-degrees angle, 200 rpm, 2.5 cmamplitude, 37° C.) were diluted in TSB until the OD₆₀₀ was 0.10, andfurther diluted 1:40 in Mueller-Hinton broth. Each well in the 384-wellassay plates was inoculated with 7.5 μl of the diluted culture. Themicroplates were placed in plastic bags and incubated without shaking at37° C. The optical density at 600 nm in the microwells was measuredafter approximately 19 hours of incubation, and the relative growthyield in each well was calculated based on the growth in the referencegroups. The MIC value was set to the highest concentration giving lessthan 30% growth in all 4 parallel wells within the sample groups. Themicroplates were further incubated for 6 hours, and optical density inthe cultures was measured once more for confirmation of the estimatedMIC-values.

Table 3 shows that APIM peptides are capable of reducing the MIC fornumerous antibiotics by at least 50% in various bacteria.

TABLE 3 APIM conc. Est MIC Strain Antibiotic μg/ml (μg/ml) E. faeciumCTC 492 Ampicillin 0 1 E. faecium CTC 492 Ampicillin 6.5 0.5 E. faeciumCTC 492 Ampicillin 13 0.5 E. faecium CTC 492 Apramycin 0 64 E. faeciumCTC 492 Apramycin 6.5 64 E. faecium CTC 492 Apramycin 13 32 E. faeciumCTC 492 Rifampicin 0 16 E. faecium CTC 492 Rifampicin 6.5 32 E. faeciumCTC 492 Rifampicin 13 4 E. faecium CTC 492 Rifampicin 20 8 E. faeciumCTC 492 Erythromycin 0 4 E. faecium CTC 492 Erythromycin 6.5 4 E.faecium CTC 492 Erythromycin 13 1 E. faecium CTC 492 Chloramphenicol 0 2E. faecium CTC 492 Chloramphenicol 6.5 2 E. faecium CTC 492Chloramphenicol 13 1 E. faecium CTC 492 Imipenem 0 4 E. faecium CTC 492Imipenem 6.5 2 E. faecium CTC 492 Imipenem 13 2 E. faecium CTC 492Tobramycin 0 64 E. faecium CTC 492 Tobramycin 6.5 64 E. faecium CTC 492Tobramycin 13 32 E. faecium CTC 492 Monensin 0 1 E. faecium CTC 492Monensin 6.5 0.5 E. faecium CTC 492 Monensin 13 0.5 E. coli NCIMB 12210Chloramphenicol 0 2 E. coli NCIMB 12210 Chloramphenicol 2 2 E. coliNCIMB 12210 Chloramphenicol 4 2 E. coli NCIMB 12210 Chloramphenicol 8 1E. coli NCIMB 12210 Novobiocin 0 64 E. coli NCIMB 12210 Novobiocin 2 32E. coli NCIMB 12210 Novobiocin 4 8 E. coli NCIMB 12210 Novobiocin 8 4 A.baumanii ATCC 19606 Rifampicin 0 2 A. baumanii ATCC 19606 Rifampicin 2 2A. baumanii ATCC 19606 Rifampicin 4 1 A. baumanii ATCC 19606 Rifampicin8 1 A. baumanii ATCC 19606 Imipenem 0 0.25 A. baumanii ATCC 19606Imipenem 2 0.125 A. baumanii ATCC 19606 Imipenem 4 0.125 A. baumaniiATCC 19606 Gentamicin 0 16 A. baumanii ATCC 19606 Gentamicin 2 16 A.baumanii ATCC 19606 Gentamicin 4 8 A. baumanii ATCC 19606 Monensin 0 64A. baumanii ATCC 19606 Monensin 2 64 A. baumanii ATCC 19606 Monensin 464 A. baumanii ATCC 19606 Monensin 8 32 A. baumanii ATCC 19606Vancomycin 0 32 A. baumanii ATCC 19606 Vancomycin 2 32 A. baumanii ATCC19606 Vancomycin 4 16 A. baumanii ATCC 19606 Novobiocin 0 8 A. baumaniiATCC 19606 Novobiocin 2 8 A. baumanii ATCC 19606 Novobiocin 4 8 A.baumanii ATCC 19606 Novobiocin 8 2

Example 8 The Effect of APIM Peptides on MDR Bacteria

The efficacy of APIM peptides as antibiotics against MDR bacteria wastested using strains of MRSA and MDR E. faecium.

Table 4 demonstrates that APIM peptides, exemplified using ATX-101, areparticularly effective against MRSA, as the MIC of APIM peptide neededto inhibit growth of two strains of MRSA was greatly reduced incomparison to a control strain of S. aureus.

TABLE 4 Starting concentration of Factor of MIC of ATX-101 StrainATX-101 (μg/ml) inhibition (μg/ml) Staphylococcus 150 1 150 aureus NCTC6571 Staphylococcus 150 0.25 37.5 aureus MRSA 1040s Staphylococcus 1500.0625 9.375 aureus MRSA 1096

In view of the anti-bacterial effect of ATX-101 on strains of MRSA, theMIC for a variety of APIM variants was determined using Staphylococcusaureus MRSA 1040s. The results in Table 5 show that all APIM peptideshave similar antibacterial activity, i.e. variation of the APIM sequencewithin the parameters defined herein, particularly at positions 3 and 4,does not reduce activity. A cell penetrating peptide (SEQ ID NO: 337)present in all of the APIM peptides, was used as a negative control.Whilst the CPP alone demonstrates some antibacterial activity, itscombination with the APIM sequence greatly improves its activity,thereby indicating that the APIM sequence is responsible for theantibacterial effect.

TABLE 5 MIC Peptide sequence APIM sequence (μg/ml) MDRWSVKWKKKRKIRRRRWSVK  32 RRRRRRRR (SEQ ID NO: 52) (SEQ ID NO: 1203) MDRWAVKWKKKRKIRRRRWAVK  32 RRRRRRRR (SEQ ID NO: 58) (SEQ ID NO: 1204) MDRWLSKWKKKRKIRRRRWLSK  32 RRRRRRRR (SEQ ID NO: 40) (SEQ ID NO: 1211) MDRWLTKWKKKRKIRRRRWLTK  32 RRRRRRRR (SEQ ID NO: 76) (SEQ ID NO: 1208) MDRWLVPWKKKRKIRRRRWLVP  32 RRRRRRRR (SEQ ID NO: 1207) (SEQ ID NO: 1212) MDRWLVKWKKKRKIRRRRWLVK  32 RRRRRRRR (SEQ ID NO: 28) (SEQ ID NO: 1198) RRRRRRRRRRR — 118(SEQ ID NO: 337)

Next, the effect of APIM peptides on the MIC of antibiotics wasdetermined using Staphylococcus aureus MRSA 1040s. Table 6 and FIG. 9show that ATX-101 reduces the MIC for erythromycin and azithromycinsignificantly. Table 7 and FIG. 10 demonstrate that similar effects areobserved for other APIM variants.

These data indicate that APIM peptides may be particularly effective intreating MRSA infections, either alone or in combination withantibiotics, particularly macrolide antibiotics.

TABLE 6 MIC (μg/ml) when combined with 10 μg/ml of MIC μg/ml ATX-101ATX-101 16 — Erythromycin >1034 2 Azithromycin >1034 8

TABLE 7 Concen- MIC of tration Eryth- of APIM romycin APIM peptide(Relative Peptide sequence sequence (μg/ml) values) MDRWLVKWKKKRKIRRRRWLVK 7.5 1000 RRRRRRRR (SEQ ID (SEQ ID NO: 1198) NO: 28)MDRWLVKWKKKRKIRRR RWLVK 15  125 RRRRRRRR (SEQ ID (SEQ ID NO: 1198)NO: 28) MDRWSVKWKKKRKIRRR RWSVK 15  100 RRRRRRRR (SEQ ID(SEQ ID NO: 1203) NO: 52) MDRWAVKWKKKRKIRRR RWAVK 15  200 RRRRRRRR(SEQ ID (SEQ ID NO: 1204) NO: 58) MDRWLSKWKKKRKIRRR RWLSK 15  200RRRRRRRR (SEQ ID (SEQ ID NO: 1211) NO: 40) MDRWLTKWKKKRKIRRR RWLTK 15 100 RRRRRRRR (SEQ ID (SEQ ID NO: 1208) NO: 76) MDRWLVPWKKKRKIRRR RWLVP15  100 RRRRRRRR (SEQ ID (SEQ ID NO: 1212) NO: 1207)

Additive effects were also observed when APIM peptides were combinedwith various antibiotics to treat a MDR strain of E. faecium (E. faeciumCCUG 37832 (TO-3)), which is commonly associated with endocarditis,urinary tract infections and infections in wounds.

The MIC for APIM peptide ATX-101 on E. faecium CCUG 37832 (TO-3) wasdetermined to be 7.5 μg/ml. Accordingly, concentrations of 8 μg/ml and16 μg/ml of ATX-101 were combined with various antibiotics selectedfrom: 2,4-Diamin, S. methizol, S. methoxa, S. dimetho, Sulfaceta,Trimeth, Flumeq, Levoflox, Pruliflox, Metronid and Nitrofur. FIG. 11shows that the MIC of each antibiotic could be reduced by combining itwith an APIM peptide. Thus, these data indicate that APIM peptides maybe particularly effective in treating E. faecium infections(particularly MDR E. faecium infections), either alone or in combinationwith antibiotics, particularly DNA gyrase inhibitors.

Example 9 Overexpression of APIM Peptides in E. coli

In order to verify further that the anti-microbial effect of the APIMpeptides arises from the APIM sequence, peptides containing only theAPIM sequence (i.e. without a cell-penetrating peptide) wereover-expressed in E. coli using the expression vector pET28. The APIMpeptide was expressed alone or as part of a fusion protein with EYFP.Expression of EYFP alone was used as a control. The expression vectorscontaining the respective peptides were transfected into the bacterialstrain E. coli BL21 (ripl). Single colonies, 4-6 of each strain, wereinoculated in 150 ml LB media (+Km/Clm) in 96 wells plates, andincubated at 37° C. Overnight cultures were diluted 1:100 and grown for1 h before induction with 1 mM IPTG (initiating peptide expression). ODwas measured every hour.

FIG. 12A shows that expression of the APIM peptide, either alone or aspart of a fusion protein with EYFP, inhibits bacterial growth. Thisresult demonstrates that the APIM sequence RWLVK (SEQ ID NO: 28) hasantibacterial properties even in the absence of a cell-penetratingpeptide.

FIG. 13 shows that APIM variant sequences are similarly effective atinhibiting bacterial growth when overexpressed in E. coli. FIG. 13Bindicates that the APIM variants RWLTK (SEQ ID NO: 76), RFSLK (SEQ IDNO: 1206) and RWLVP (SEQ ID NO: 1207) are particularly effective.However, all of the variants tested show a significant inhibitory effecton bacterial growth.

Example 10 Determination of APIM Peptide Interaction with the β-ClampProtein from E. coli

Microscale thermophoresis (MST) was used to determine the dissociationconstant for various APIM containing peptides.

The β-clamp protein from E. coli was labeled with a fluorescentmolecule. Concentration of PCNA was kept constant, whereas dilutions ofeach APIM containing peptide were prepared (1:1). In a mix of proteinand peptide, the signal was recorded in all capillaries with varyingconcentrations of the unlabeled peptide, and any change ofthermophoretic properties was observed as a change in fluorescenceintensity.

Table 8 shows that various APIM peptides show specific interactions withthe β-clamp protein (a low Kd value indicates a strong interaction,whereas a high Kd value indicates a weak interaction). The R11 peptide(SEQ ID NO: 337) was used as a control and no data could be obtained byMST for this peptide, indicating that this peptide does not interactwith the β-clamp protein. This data further verifies that the APIMsequence contributes to the antibacterial effects of the APIM peptides.

TABLE 8 APIM Peptide sequence sequence Kd MDRWLVKWKKKRKIRRRRRRRRRRRRWLVK 101 (SEQ ID NO: 1198) (SEQ ID NO: 28) MDRWSVKWKKKRKIRRRRRRRRRRRRWSVK 408 (SEQ ID NO: 1203) (SEQ ID NO: 52) MDRWAVKWKKKRKIRRRRRRRRRRRRWAVK  57 (SEQ ID NO: 1204) (SEQ ID NO: 58) MDRWLSKWKKKRKIRRRRRRRRRRRRWLSK 115 (SEQ ID NO: 1211) (SEQ ID NO: 40) MDRWLTKWKKKRKIRRRRRRRRRRRRWLTK  22 (SEQ ID NO: 1208) (SEQ ID NO: 76) MDRWLVPWKKKRKIRRRRRRRRRRRRWLVP 366 (SEQ ID NO: 1212) (SEQ ID NO: 1207) MDRFLSKWKKKRKIRRRRRRRRRRRRFLSK 511 (SEQ ID NO: 1210) (SEQ ID NO: 41) MDRFSLKWKKKRKIRRRRRRRRRRRRFSLK  20 (SEQ ID NO: 1209) (SEQ ID NO: 1206) R11 (SEQ ID NO: 337) — Nd

Example 11 Determination of the Effect of UV Radiation on theAnti-Microbial Properties of APIM Peptides

Pre-cultures of E. coli BL21 (ripl) were grown over-night in LB at 37°C. The cultures were then diluted 1:100 and 150 ml/well was added/wellin 96 wells plates. The cultures were further incubated for 1 h and eachplate was exposed to UVC, 2 J/cm², with a Stratalinker. The plates wereincubated for 30 minutes following UV treatment and various APIMpeptides, 15 μM or 30 μM of each peptide, were added to 6 parallelwells. OD₆₆₀ was measured every hour. Values were normalized and averagewas plotted.

FIGS. 14A and B show the effect of various APIM peptides (30 μM) onbacteria that have not been exposed to UV radiation. The graph in FIG.14B shows the same growth curves as FIG. 14A without the controls (nopeptide was added), which are the two highest grow curves in FIG. 14A.Thus, FIG. 14B shows that there is some re-growth 5 hours after the APIMpeptides were added. However, this assay shows that all of the APIMpeptides tested have a significant inhibitory effect on bacterialgrowth.

FIGS. 15A and B are equivalent to FIGS. 14A and B when the bacteria havebeen exposed to UV radiation. These Figures demonstrate that there is nore-growth when the APIM peptides are combined with UV radiation.Furthermore, no re-growth was observed in samples even when they wereincubated over-night (data not shown). This suggests that UV radiationsensitizes the cells to APIM peptides or vice versa. Thus, these datademonstrate that a combination of APIM peptides and UV radiation,particularly UVC radiation, may be useful in treating bacterialinfections.

The treatments using APIM peptides at 15 μM (data not shown) showedsimilar effects to the 30 μM treatment, which is shown in FIGS. 14 and15.

FIG. 16 shows that treatment with UV radiation is effective even whenusing lower concentrations of APIM peptides. APIM peptide concentrationsof 15 μM (FIG. 16A), 7.5 μM (FIG. 16B) and 3.75 μM (FIG. 16C) were alleffective at inhibiting bacterial growth following treatment with UVradiation. Overall, these data show that bacteria are more 2-5 fold moresensitive to APIM peptides after UV-irradiation.

Example 12 Effect of ATX-101 on Methicillin Resistant StaphylococcusAureus (MRSA) Biofilm Under Flow

APIM peptides were tested to determine whether they have an effect onbiofilm formation.

The IBIDI flow-system coupled with EVOS Auto Imaging system wasoptimized and used for testing the effect of ATX-101 on MRSA biofilmunder flow. MRSA 1040 (u50) was used as model organism; it normallyproduces a dense biofilm in the growth channel during 36 hours of flow.The effect of 3 different concentrations of ATX-101 was tested (7 μg/ml,3.5 μg/ml and 0.8 μg/ml). The flow system was programmed with sharestress similar to those found in capillary networks (3.49 dyne/cm²), 2%Luria Bertani (LB) was used for dilution and flow medium. Good effect ofATX-101 was observed for all tested concentrations, the highest andlowest concentrations are presented in FIGS. 17 and 18, respectively.

Example 13 Determining the Minimum Inhibitory Concentration (MIC) ofVarious APIM Peptides on Bacteria

The MICs for various APIM peptides were determined for various bacteria,as described above. The results are shown in Table 9 and show that allof the tested variants have anti-bacterial properties, when used alone,across a variety of bacteria. The results also demonstrate that the APIMpeptides are particularly effective against MDR bacteria, e.g. E.faecium TO-3 and MRSA 1040.

TABLE 9 MIC (μM) E.faecium E.faecium MRSA S.aureus Peptide sequenceP.aeruginosa E.coli A.baumanii TO-3 TO-12 1040 NCTC6571MDRWLVKWKKKRKIRRRRRRRRRRR 37.9 nd nd 7.5 16.8 7.5 37.9 (SEQ ID NO: 1198)MDRWSVKWKKKRKIRRRRRRRRRRR nd 37.9 nd 7.5 37.9 7.5 37.9 (SEQ ID NO: 1203)MDRWAVKWKKKRKIRRRRRRRRRRR nd nd nd 7.5 25.3 7.5 37.9 (SEQ ID NO: 1204)MDRWLSKWKKKRKIRRRRRRRRRRR nd 37.9 nd 7.5 16.8 7.5 37.9 (SEQ ID NO: 1211)MDRWLTKWKKKRKIRRRRRRRRRRR nd nd nd 7.5 25.3 7.5 37.9 (SEQ ID NO: 1208)MDRWLVPWKKKRKIRRRRRRRRRRR nd 37.9 25.3 7.5 25.3 7.5 37.9(SEQ ID NO: 1212) MDRFLSKWKKKRKIRRRRRRRRRRR nd 37.9 nd 7.5 25.3 7.5 37.9(SEQ ID NO: 1210) MDRFSLKWKKKRKIRRRRRRRRRRR nd nd nd 7.5 25.3 7.5 37.9(SEQ ID NO: 1209)

1. An agent, or a composition containing an agent, for use in treatingor preventing a bacterial infection in a subject, wherein said agentcomprises: (i) an oligopeptidic compound comprising a PCNA interactingmotif and a domain that facilitates the cellular uptake of saidcompound, wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ IDNO: 1) and wherein: X₁ is a basic amino acid; X₂ is an aromatic aminoacid; X₃ is an uncharged amino acid other than an aromatic amino acid,Glycine (G) and Proline (P); X₄ is any amino acid other than Proline(P), an acidic amino acid or an aromatic amino acid; and X₅ is a basicamino acid or Proline (P); or (ii) a nucleic acid molecule comprising asequence encoding the oligopeptidic compound of (i).
 2. Use of an agentin the manufacture of a medicament for the treatment or prevention of abacterial infection in a subject, wherein said agent comprises: (i) anoligopeptidic compound comprising a PCNA interacting motif and a domainthat facilitates the cellular uptake of said compound, wherein the PCNAinteracting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) and wherein: X₁ is abasic amino acid; X₂ is an aromatic amino acid; X₃ is an uncharged aminoacid other than an aromatic amino acid, Glycine (G) and Proline (P); X₄is any amino acid other than Proline (P), an acidic amino acid or anaromatic amino acid; and X₅ is a basic amino acid or Proline (P); or(ii) a nucleic acid molecule comprising a sequence encoding theoligopeptidic compound of (i).
 3. A method of treating or preventing abacterial infection, said method comprising administering an agent, or acomposition containing an agent, to a subject in need thereof, whereinsaid agent comprises: (i) an oligopeptidic compound comprising a PCNAinteracting motif and a domain that facilitates the cellular uptake ofsaid compound, wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ IDNO: 1) and wherein: X₁ is a basic amino acid; X₂ is an aromatic aminoacid; X₃ is an uncharged amino acid other than an aromatic amino acid,Glycine (G) and Proline (P); X₄ is any amino acid other than Proline(P), an acidic amino acid or an aromatic amino acid; and X₅ is a basicamino acid or Proline (P); or (ii) a nucleic acid molecule comprising asequence encoding the oligopeptidic compound of (i).
 4. The agent,composition, use or method of any one of claims 1 to 3, wherein saidagent or composition is provided as a combined preparation with one ormore additional active agents for separate, simultaneous or sequentialuse or administration.
 5. A kit or product containing an agent orcomposition as defined in claim 1 and one or more additional activeagents as a combined preparation for simultaneous, sequential orseparate use in the treatment or prevention of a bacterial infection. 6.An agent, or a composition containing an agent, for use as a sensitizerfor UV radiotherapy in the treatment or prevention of a bacterialinfection in a subject, or in the treatment or prevention of a bacterialinfection in subject which involves UV radiotherapy, wherein said agentcomprises: (i) an oligopeptidic compound comprising a PCNA interactingmotif and a domain that facilitates the cellular uptake of saidcompound, wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ IDNO: 1) and wherein: X₁ is a basic amino acid; X₂ is an aromatic aminoacid; X₃ is an uncharged amino acid other than an aromatic amino acid,Glycine (G) and Proline (P); X₄ is any amino acid other than Proline(P), an acidic amino acid or an aromatic amino acid; and X₅ is a basicamino acid or Proline (P); or (ii) a nucleic acid molecule comprising asequence encoding the oligopeptidic compound of (i).
 7. The method ofclaim 3 or 4, wherein said method further comprises a step of UVradiotherapy, which may be administered simultaneously, sequentially orseparately to said agent or composition.
 8. An in vitro method of: (i)preventing, inhibiting or reducing bacterial colonisation and/or growthin or on a surface, product or material; or (ii) preventing, inhibitingor reducing unwanted or undesirable bacterial colonisation and/or growthof a bacterial cell, comprising administering an agent or composition asdefined in claim 1 to a surface, product or material susceptible tobacterial growth or a bacterial cell or a cell culture, optionallysimultaneously, sequentially or separately administering one or moreadditional active agents to said surface, product or material, cell orcell culture.
 9. The method of claim 8, wherein said method comprises astep of exposing said surface, product or material to UV radiation,prior to, contemporaneously with or after administering said agent orcomposition.
 10. The agent, composition, use, method, kit or product ofany one of claims 1 to 7 wherein said bacterial infection comprises abiofilm or the in vitro method of claim 8 or 9, wherein said bacterialcolonisation and/or growth is a biofilm.
 11. The agent, composition,use, method, kit or product of any one of claims 1 to 7 or in vitromethod of any one of claims 8 to 10, wherein the bacterial infection iscaused by, or the bacterial cell is, a bacterium selected from any ofthe genera Achromobacter, Acinetobacter, Actinobacillus, Aeromonas,Agrobacterium, Alcaligenes, Alteromonas, Bacteroides, Bartonella,Borrelia, Bordetella, Brucella, Burkholderia, Campylobacter,Cardiobacterium, Chlamydia, Chlamydophila, Chromobacterium,Chyseobacterium, Chryseomonas, Citrobacter, Clostridium, Comamonas,Corynebacterium, Coxiella, Cryptobacterium, Edwardsiella, Eikenella,Enterobacter, Enterococcus, Erwinia, Helicobacter, Kingella, Klebsiella,Lactobacillus, Lactococcus, Legionella, Leptospira, Leptotrichia,Leuconostoc, Listeria, Listonella, Mobiluncus, Moraxella, Morganella,Mycobacterium, Mycoplasma, Neisseria, Nocardia, Nocardiopsis, Pantoea,Parachlamydia, Pasteurella, Peptococcus, Peptostreptococcus, Prevotella,Propionibacterium, Proteus, Providencia, Pseudomonas, Ralstonia,Rickettsia, Salmonella, Shewenella, Shigella, Sphingobacterium,Sphingomonas, Staphylococcus, Stenotrophomonas, Streptobacillus,Streptococcus, Streptomyces, Treponem and Yersinia.
 12. The agent,composition, use, method, kit or product of any one of claim 1 to 7 or11 or in vitro method of any one of claims 8 to 11, wherein thebacterium is a MDR bacterium.
 13. The agent, composition, use, method,kit, product or in vitro method of claim 12, wherein the MDR bacteriumis a Methicillin-resistant Staphylococcus aureus (MRSA) bacterium or anEnterococcus faecium bacterium.
 14. The agent, composition, use, method,kit or product of any one of claim 1 to 7, 11 or 12, wherein the subjecthas bacterial infectious disease selected from bacterial pneumonia,gastric ulcers, bacterial meningitis, Legionellosis, Pertussis,Salmonellosis, Tuberculosis or sepsis.
 15. The agent, composition, use,method, kit or product of any one of claim 1 to 7, 11 or 12, wherein thesubject has cystic fibrosis.
 16. The agent, composition, use, method,kit or product of any one of claims 1 to 7 or 11 to 15, wherein theinfection is a respiratory infection, stomach infection, GIT infection,blood infection, skin infection, bladder infection, kidney infection,ear infection, eye infection or meningial infection.
 17. The agent,composition, use, method, kit, product or in vitro method of any one ofclaims 4 to 16, wherein said one or more additional active agents is anantibiotic.
 18. The agent, composition, use, method, kit, product or invitro method of claim 17, wherein said antibiotic agent is selected fromone or more of a Macrolide, an Aminocoumarin, an Aminoglycosid, anAnsamycin, a Carbapenem, a Cephalosporin, a Glycopeptide, a Lincosamide,a Lipopeptide, a Monobactam, a Nitrofuran, an Oxazolidonone, aPenicillin, a Penicillin combination, a Polyether antibiotic, aPolypeptide antibiotic, a Quinolone, a sulfonamide, a Tetracycline oranother antibiotic.
 19. A product, material, device or implant which iscoated, impregnated or chemically bonded with an agent or composition asdefined in claim 1, wherein when the product, material, device orimplant is impregnated: (i) said product or material is a bandage,gauze, surgical tape, cotton swab, puff, fleece, sponge or supportivematrix, diaper, glove, sock, contact lens or contact lens storage case;or (ii) said device or implant is a stent, an ear tube, an artificialeye lens, an orthopedic implant, an artificial bone, a dental implant, acardiac device, a cosmetic implant, an intra-uterine device, a catheteror a prosthetic device.
 20. A method of producing product, material,device or implant which is coated, impregnated or chemically bonded withan agent or composition as defined in claim 1, comprising: (i) providinga product, material, device or implant; and (ii) coating or impregnatingsaid device with said agent or composition, or chemically bonding saidagent or composition to said product, material, device or implant,wherein when the product, material, device or implant is impregnated:(i) said product or material is a bandage, gauze, surgical tape, cottonswab, puff, fleece, sponge, supportive matrix, diaper, glove, sock,contact lens or contact lens storage case; or (ii) said device orimplant is a stent, an ear tube, an artificial eye lens, an orthopedicimplant, an artificial bone, a dental implant, a cardiac device, acosmetic implant, an intra-uterine device, a catheter or a prostheticdevice.
 21. A method of preventing or inhibiting the formation of abacterial biofilm on a product, material, device or implant, said methodcomprising: (i) providing a product, material, device or implant; and(ii) coating or impregnating said device with an agent or composition asdefined in claim 1, or chemically bonding an agent or composition asdefined in claim 1 to said product, material, device or implant.
 22. Themethod of claim 21, wherein bacteria is as defined in any one of claims11 to
 13. 23. The in vitro method of any one of claims 8 to 13 orproduct or material of claim 19 or method of any one of claims 20 to 22,wherein said product or material is a bandage, plaster, gauze, surgicaltape, cotton swab, puff, fleece, sponge, supportive matrix, wounddressing, diaper, glove, sock, contact lens or contact lens storagecase.
 24. The device or implant of claim 19 or method of any one ofclaims 20 to 22, wherein said device or implant is a medical or surgicaldevice or implant.
 25. The method of claim 24, wherein the device orimplant is selected from a stent, an ear tube, an artificial eye lens,an orthopedic implant, an artificial bone, a dental implant, a cardiacdevice, a cosmetic implant, an intra-uterine device, a catheter or aprosthetic device.
 26. The agent, composition, use, method, kit,product, material, device, implant or in vitro method of any one ofclaims 1 to 25, wherein X₃ is a hydrophobic or polar amino acid.
 27. Theagent, composition, use, method, kit, product, material, device, implantor in vitro method of any one of claims 1 to 25, wherein X₃ is ahydrophobic amino acid.
 28. The agent, composition, use, method, kit,product, material, device, implant or in vitro method of any one ofclaims 1 to 26, wherein X₃ and/or X₄ is a polar amino acid.
 29. Theagent, composition, use, method, kit, product, material, device, implantor in vitro method of any one of claims 1 to 26, wherein one of X₃ andX₄ is a polar amino acid.
 30. The agent, composition, use, method, kit,product, material, device, implant or in vitro method of claim 28 or 29,wherein the polar amino acid is serine (S) or threonine (T).
 31. Theagent, composition, use, method, kit, product, material, device, implantor in vitro method of any one of claims 1 to 26, wherein X₄ and/or X₅ isa basic amino acid.
 32. The agent, composition, use, method, kit,product, material, device, implant or in vitro method of any one ofclaims 1 to 26, wherein X₅ is a basic amino acid.
 33. The agent,composition, use, method, kit, product, material, device, implant or invitro method of any one of claims 1 to 26, wherein X₄ is a hydrophobicamino acid.
 34. The agent, composition, use, method, kit, product,material, device, implant or in vitro method of any one of claim 1 to25, 31 or 32, wherein the basic amino acid selected from any one ofarginine (R), lysine (K), histidine (H), ornithine (Orn), methyllysine(MeK), diaminobutyric acid (Dbu), citrulline (Cit), acetyllysine (AcK),and any suitable basic amino acid selected from the non-conventionalamino acids in Table
 2. 35. The agent, composition, use, method, kit,product, material, device, implant or in vitro method of any one ofclaims 1 to 25, wherein X₃ is selected from any one of leucine (L),isoleucine (I), valine (V), alanine (A) methionine (M), norleucine(Nor), serine (S) or threonine (T), glutamine (Q), aspargine (N) orcysteine (C) or any suitable hydrophobic or polar amino acid selectedfrom the non-conventional amino acids in Table
 2. 36. The agent,composition, use, method, kit, product, material, device, implant or invitro method of any one of claims 1 to 25, wherein X₄ is selected fromany one of from V, L, I, A, M, Nor, S, T, Q, N, H, K, R, G or C or anysuitable hydrophobic, polar, basic or thiol-containing amino acidselected from the non-conventional amino acids in Table
 2. 37. Theagent, composition, use, method, kit, product, material, device, implantor in vitro method of any one of claims 1 to 25, wherein the aromaticamino acid is selected from any one of tryptophan (W), tyrosine (Y),phenylalanine (F), tert.-butylglycine, cyclohexylalanine,tert.-butylphenylalanine, biphenylalanine or tri tert.-butyltryptophanor any suitable aromatic amino acid selected from the non-conventionalamino acids in Table
 2. 38. The agent, composition, use, method, kit,product, material, device, implant or in vitro method of any one ofclaims 1 to 25, wherein the aromatic amino acid is selected from any oneof tryptophan (W), tyrosine (Y) or phenylalanine (F).
 39. The agent,composition, use, method, kit, product, material, device, implant or invitro method of any one of claims 1 to 39, wherein the PCNA interactingmotif comprises a sequence selected from: (SEQ ID NO: 2)[R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T/N/Q/C]-[V/L/I/A/M/G/S/T/N/Q/R/H/K/C]-[K/R/H/P]; (SEQ ID NO: 3)[R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T/N/Q]-[V/L/I/A/M/G/S/T/N/Q/R/H/K]-[K/R/H/P]; (SEQ ID NO: 4)[R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]-[V/L/I/A/M/G/S/ T/N/Q/R/H/K]-[K/R/H/P];(SEQ ID NO: 5) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]-[V/L/I/A/M/G/S/T/N/Q/R/H/K]-[K/R/H]; (SEQ ID NO: 6)[R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]-[V/L/I/A/M/G/S/ T/R/K]-[K/R/H];(SEQ ID NO: 7) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]-[V/L/I/A/M/G/S/T]-[K/R/H]; (SEQ ID NO: 8)[R/K]-[W/F/Y]-[L/I/V/A/M/S/T]-[V/L/I/A/M/G/S/T]- [K/R]; (SEQ ID NO: 9)[R/K]-[W/F/]-[L/I/V/A/M/S/T]-[V/L/I/A/M/G/S/T]- [K/R]; (SEQ ID NO: 10)[R/K]-[W/F]-[L/I/V/A/M/T]-[V/L/I/A/M/G/S/T]- [K/R]; (SEQ ID NO: 11)[R/K]-[W/F]-[L/I/V/A/M/T]-[V/L/I/A/M/S/T]-[K/R]; (SEQ ID NO: 12)[R/K]-[W/F]-[L/I/V/A/M/S/T]-[V/L/I/A/M/G]-[K/R]; (SEQ ID NO: 13)[R/K]-[W/F]-[L/I/A/V/M/T]-[V/L/I/A/M/S/T]-[K/R]; (SEQ ID NO: 14)[R/K]-[W/F]-[L/I/V/A/M/S/T]-[V/L/A/I/S/T]-[K/R]; (SEQ ID NO: 15)[R/K]-[W/F]-[L/V/I/A/T]-[V/L/A/I/S/T]-[K/R]; (SEQ ID NO: 16)[R]-[W/F/Y]-[L/V/I/A]-[V/L/A/S/T/M]-[K/R]; (SEQ ID NO: 17)[R]-[W/F/Y]-[L/V/I/A/T]-[V/L/A/S/T/M]-[K]; (SEQ ID NO: 18)[R/K]-[F/Y]-[L/V/I/A]-[V/L/A/I/M]-[K/R]; (SEQ ID NO: 19)[R/K]-[W/F/Y]-[L/I/V/A]-[V/L/I/A]-[K/R]; (SEQ ID NO: 20)[R/K]-[W/Y]-[L/V/I/A/S/T]-[V/L/A/S/T/M]-[K/R]; or (SEQ ID NO: 21)[K]-[F/Y/W]-[I/L/V/A/T]-[V/L/A/I/S/T/M]-[K].


40. The agent, composition, use, method, kit, product, material, device,implant or in vitro method of any one of claims 1 to 39, wherein X₁ andX₂ are RW, RF, KF, KW, RY or KY.
 41. The agent, composition, use,method, kit, product, material, device, implant or in vitro method ofany one of claims 1 to 40, wherein X₃ and X₄ are LV, LL, LA, AL, VL, VI,LI, IL, VV, VA, IV, II, AV, IA, AI, AM, LM, LS, LT, IS, MV, TV, AA, IM,LN, LQ, VM, TL, SL, IT, VT, LG, MA, ML, NL, QL, QI, TI, SI, AS, VS, SV,CA, IG, LR, VR, TK or IR.
 42. The agent, composition, use, method, kit,product, material, device, implant or in vitro method of any one ofclaims 1 to 41, wherein X₃ and X₄ are not AG, AC, CC, NN, QQ, NQ, QN,TS, SS, ST or TT.
 43. The agent, composition, use, method, kit, product,material, device, implant or in vitro method of any one of claims 1 to42, wherein the PCNA interacting motif comprises a sequence selectedfrom any one of SEQ ID NOs: 28, 22 to 27, 29 to 297, 1206 or
 1207. 44.The agent, composition, use, method, kit, product, material, device,implant or in vitro method of any one of claims 1 to 43, wherein thedomain that facilitates the cellular uptake of the oligopeptidiccompound is a cell penetrating peptide (CPP).
 45. The agent,composition, use, method, kit, product, material, device, implant or invitro method of claim 44, wherein the CPP is selected from any one of:(i) an antennapedia class peptide; (ii) a protegrin class peptide; (iii)a HIV-TAT class peptide; (iv) an amphipathic class peptide selected froman amphipathic and net positively charged peptide, a proline-richamphipathic peptide, a peptide based on the Pep-1 peptide and a peptidebased on the MPG peptide; (v) a peptide exhibiting high α-helicalcontent; (vi) a peptide comprising oligomers of basic amino acids; (vii)pVEC; (viii) a calcitonin-derived peptide; and (ix) an amphiphiliccyclic CPP.
 46. The agent, composition, use, method, kit, product,material, device, implant or in vitro method of claim 44 or 45, whereinthe CPP is selected from any one of SEQ ID NOs: 337, 302 to 336, 338 to1162, or 1213 to 1223 or a fragment and/or derivative thereof.
 47. Theagent, composition, use, method, kit, product, material, device, implantor in vitro method of any one of claims 1 to 46, wherein the agentfurther comprises a linker domain.
 48. The agent, composition, use,method, kit, product, material, device, implant or in vitro method ofclaim 47, wherein the linker domain comprises a nuclear localisationsignal sequence.
 49. The agent, composition, use, method, kit, product,material, device, implant or in vitro method of claim 48, wherein thelinker domain or nuclear localisation signal sequence comprises asequence selected from any one of: (i) a peptide of 4-20 amino acids,wherein at least 4 amino acids are positively charged amino acids,preferably selected from K, R or H; and/or (ii) a sequence selected fromany one of SEQ ID NOs: 1176, 1162 to 1175 or 1177 to 1181 or a fragmentand/or derivative thereof.
 50. The agent, composition, use, method, kit,product, material, device, implant or in vitro method of claim 48 or 49,wherein the linker domain or nuclear localisation signal sequencecomprises a sequence selected from any one of SEQ ID NOs: 1162 to 1181or a fragment and/or derivative thereof, preferably wherein saidfragment and/or derivative comprises at least 4 positively charged aminoacids, preferably selected from K, R or H.
 51. The agent, composition,use, method, kit, product, material, device, implant or in vitro methodof any one of claims 1 to 50, wherein the agent comprises a PCNAinteracting motif as set forth in SEQ ID NO: 28, a linker or nuclearlocalisation signal sequence as set forth in SEQ ID NO: 1176 and a cellpenetrating signal sequence as set forth in SEQ ID NO:
 337. 52. Theagent, composition, use, method, kit, product, material, device, implantor in vitro method of any one of claims 1 to 51, wherein the agentcomprises a sequence as set forth in any one of SEQ ID NOs: 1198, 1182to 1197, 1199 to 1204 or 1208 to 1212, preferably wherein the agentcomprises a sequence as set forth in SEQ ID NO: 1198, 1203, 1204 or 1208to 1212.